Polypeptides

ABSTRACT

Disclosed are a polypeptide (including that in soluble form) as receptor for a novel cytokine, i.e., interleukin-18, a DNA encoding the polypeptide, and the uses of the polypeptide including pharmaceutical and neutralizer to interleukin-18. Pharmaceuticals with the polypeptide is useful to treat and prevent autoimmune and allergic disease because it suppresses and regulates excessive immunoreaction.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention relates to a novel receptor protein whichrecognizes a cytokine, more particularly, to a novel polypeptide whichrecognizes interleukin-18 (hereinafter abbreviated as “IL-18”).

[0003] 2. Description of the Prior Art

[0004] IL-18 is a type of cytokine or substance which mediates signaltransduction in immune system. As seen in Japanese Patent Kokai Nos.27,189/96 and 193,098/96 and Haruki Okamura et al., Nature, Vol. 378,No. 6,552, pp. 88-91 (1995), IL-18 was provisionally designated as“interferon-gamma inducing factor” immediately after its discovery: Thisdesignation was changed later into “IL-18” in accordance with theproposal in Shimpei Ushio et al., The Journal of Immunology, Vol. 156,pp. 4,274-4,279 (1996). IL-18 in mature form consists of 157 amino acidsand possesses properties of inducing in immunocompetent cells theproduction of interferon-gamma (hereinafter abbreviated as “IFN-γ”)which is known as useful biologically-active protein, as well as ofinducing and enhancing the generation and cytotoxicity of killer cells.Energetic studies are now in progress to develop and realize varioususes of IL-18 in pharmaceuticals such as antiviral, antimicrobial,antitumor and anti-immunopathic agents which have been in greatexpectation because of these properties of IL-18.

[0005] As described above, in nature, cytokines including IL-18 areproduced and secreted as substances responsible for signal transductionin immune system. Therefore, excessive amounts of cytokines may disturbthe equilibria in immune system when they are produced or administeredin the body of mammals. The surface of usual mammalian-cells may bearcertain sites or “receptors” which are responsible for recognition ofcytokines: Secreted cytokines transduce no signal in cells till they arebound to the receptors. In normal immune system, there would be definiteequilibria between respective cytokines and their receptors. Thus, inthis field, with the purpose of developing and realizing IL-18 aspharmaceuticals, in addition to the clarification of physiologicalactivities of IL-18, an expedited establishment of mass production andcharacterization of IL-18 receptor (hereinafter abbreviated as “IL-18R”)have been in great expectation.

SUMMARY OF THE INVENTION

[0006] In view of the foregoing, the first object of this invention isto provide a polypeptide as IL-18R which can be easily prepared on alarge scale.

[0007] The second object of this invention is to provide uses of suchpolypeptide as pharmaceuticals.

[0008] The third object of this invention is to provide a DNA whichencodes the polypeptide.

[0009] The fourth object of this invention is to provide a process toprepare the polypeptide.

[0010] The fifth object of this invention is to provide an agent toneutralize IL-18 using the polypeptide.

[0011] The sixth object of this invention is to provide a method toneutralize IL-18 using the polypeptide.

[0012] We energetically and extensively screened various means whichmight attain these objects, eventually resulting in the finding that asubstance which recognized IL-18 was present in L428 cell, a type oflymphoblastoid cell derived from a patient with Hodgkin's disease. Weisolated and characterized this substance, revealing that its nature wasproteinaceous, as well as that it well recognized and bound IL-18 evenwhen in isolated form. It was also found that the IL-18R thus identifiedwas efficacious in treatment and prevention of various diseasesresulting from excessive immunoreaction, such as autoimmune diseases,because in mammals including human, IL-18R recognized and neutralizedIL-18 which activated immune system. Further, we have energeticallystudied L428 cell using as probe some partial amino acid sequences ofthe IL-18R, resulting in obtainment of a DNA which did encode IL-18R. Weconfirmed that a polypeptide obtained by bringing such DNAs intoexpression in artificial manner well recognized IL-18 and shared someessential physiological activities with the IL-18R separated from L428cell, as well as that it was preparable in desired amounts byrecombinant DNA techniques using such DNA. Thus we accomplished thisinvention.

[0013] More particularly, this invention attains the first object with apolypeptide as IL-18R, which is obtainable through gene expression.

[0014] This invention attains the second object with an agent for IL-18Rsusceptive diseases, which contains as effective ingredient suchpolypeptide.

[0015] This invention attains the third object with a DNA which encodesthe polypeptide.

[0016] This invention attains the forth object with a process to preparepolypeptide, comprising bringing into expression a DNA which encodes thepolypeptide, and collecting the resultant polypeptide.

[0017] This invention attains the fifth object with an agent toneutralize IL-18, which contains as effective ingredient thepolypeptide.

[0018] This invention attains the sixth object with a method toneutralize IL-18, characterized by allowing the polypeptide to act onIL-18.

[0019] L428 cell, which is feasible in this invention, have beendeposited in the Patent Microorganism Depository, National Institute ofBioscience and Human-Technology, Agency of Industrial Science andTechnology, 1-3, Higashi 1 chome, Tsukuba-shi, Ibaraki-ken, 305, Japan,under the accession number of “FERM BP-5777” on and after Dec. 24, 1996.

BRIEF EXPLANATION OF THE ACCOMPANYING DRAWINGS

[0020]FIG. 1 shows that the monoclonal antibody MAb #117-10C binds toL428 cells and IL-18R while competing with IL-18.

[0021]FIG. 2 is an image of intermediate tone given on display, whichshows IL-18R on gel electrophoresis visualized by the Western blottingmethod using the monoclonal antibody MAb #117-10C.

[0022]FIG. 3 shows the inhibitory action of the monoclonal antibody MAb#117-10C on the activity of IL-18.

[0023]FIG. 4 is the chromatogram obtained by applying to IL-18R animmunoaffinity chromatography using the monoclonal antibody MAb#117-10C.

[0024]FIG. 5 is the peptide map of IL-18R.

[0025]FIG. 6 shows, the structure of the recombinant DNA“pcDNA/HuIL-18R” of this invention.

[0026]FIG. 7 shows the structure of the recombinant DNA “pEFHIL18R-14”of this invention.

[0027]FIG. 8 shows the structure of the recombinant DNA“pEFHIL18RD1-2-H” of this invention.

[0028]FIG. 9 shows the structure of the recombinant DNA “pEFHIL18RD1-H”of this invention.

[0029]FIG. 10 shows the structure of the recombinant DNA “pEFMIL18RSHT”of this invention.

[0030] Throughout the Figures, the symbol “Pcmv” indicates thecytomegalo virus promotor; “EF1αP”, the elongation factor promotor;“IL-18R cDNA”, the cDNA encoding the polypeptide of this invention;“EFHIL18R-14 cDNA”, the cDNA encoding the soluble polypeptide of humanorigin according to this invention; “HIL18RD1-2-H cDNA”, the cDNAencoding the soluble polypeptide of human origin according to thisinvention; “HIL18RD1-H cDNA”, the cDNA encoding the soluble polypeptideof human origin according to this invention; and “EFMIL18RSHT cDNA”, thecDNA encoding the soluble polypeptide of mouse origin according to thisinvention.

DETAILED DESCRIPTION OF THE INVENTION

[0031] This invention relates to a polypeptide as IL-18R, which isobtainable through gene expression. The polypeptide of human originaccording to this invention usually contains as partial amino acidsequence(s) one or more amino acid sequences of SEQ ID NOs: 12 to 19: Asa whole; it contains a part or whole of the amino acid sequence of SEQID NO: 20. While the polypeptide of mouse origin according to thisinvention usually contains a part or whole of the amino acid sequence ofSEQ ID NO: 21. Thus, the wording “polypeptide” as referred to in thisinvention shall include, in addition to those which wholly contain theamino-acid sequence of either SEQ ID NO: 20 or 21, for example, thosewhich contain the same amino acid sequence but with addition of one ormore amino acids, in particular, those which contain one or more aminoacids linked to the C- and/or N-termini in SEQ ID NO: 20 or 21; thosewhich contain the same amino acid sequence as in SEQ ID NOs: 20 and 21but with deletion of one or more amino acids, in particular, solublepolypeptides which contain the amino acid sequences of SEQ ID NOs: 22 to25; and those which contain either of the amino acid sequences asdescribed above but with a saccharide chain, as far as they areobtainable through gene expression and possess the essential functionsof IL-18R. As to IL-18, those of human and mouse origins commonlyconsisting of 157 amino acids have been documented: Human IL-18 bearsthe amino acid sequence of SEQ ID NO: 26 (where the amino acid withsymbol “Xaa” represents either isoleucine or threonine), while mousecounterpart, the amino acid sequence of SEQ ID NO: 27 (where the aminoacid with symbol “Xaa” represents either methionine or threonine).

[0032] The polypeptide of this invention is usually prepared by applyingrecombinant DNA techniques, more particularly, by bringing intoexpression in artificial manner a DNA which encodes the polypeptide, andcollecting the resultant polypeptide. This invention provides, inaddition to a DNA which encodes the polypeptide, a process to preparethe polypeptide using recombinant DNA techniques: By practicing such aprocess according to this invention, desired amounts the polypeptide canbe easily obtained.

[0033] The DNA which is used in this invention are those whichoriginating natural sources, those which can be obtained by artificiallymodifying them and those which can be obtained through chemicalsynthesis, provided that they do encode the polypeptide. Generally, inthis field, in case of artificially expressing DNAs which encodepolypeptides, one may replace one or more nucleotides in the DNAs withdifferent nucleotides and/or link an appropriate nucleotide sequence tothe DNAs, with purpose of improving their expression efficiency and/orthe physiological and physicochemical properties of the polypeptides.Such modifications are feasible in the DNA of this invention of course:For example, one can link to the 5′- and 3′-termini of the DNA asdescribed above recognition sites for appropriate restriction enzymes,initiation and termination codons, promoters and/or enhancers, as far asthe final polypeptide products do retain desired physiologicalactivities. Thus, the wording “DNA” as referred to in this inventionshall mean, in addition to those which encode any polypeptides asdescribed above, those which are complementary thereto, and furtherthose where one or more nucleotides have been replaced with differentnucleotides while conserving the amino acid sequence.

[0034] To obtain such a DNA from natural sources, for example, mammaliancells including epithelial cells, endothelial cells, interstitial cells,chondrocytes, monocytes, granulocytes, lymphocytes, neurocytes and theirestablished cell lines of human and mouse origins are screened witholigonucleotides as probe which can be prepared with reference to theamino acid sequences of SEQ ID NOs: 12 to 25. Examples of preferredcells are cell lines which are obtained by establishing hemopoieticcells including lymphocytes, in particular, JM cells, HDLM-2 cells,MOLT-16 cells and PEER cells described in Jun Minowada, Cancer Review,Vol. 10, pp. 1-18 (1988), and lymphoblastoid cells such as L428 cell(FERM BP-5777), KG-1 cell (ATCC CCL-246) and U-937 cells (ATCCCRL-1593). The human and mouse DNAs obtained in this way usually containa part or whole of respective nucleotide sequences of SEQ ID NOs: 1 and2. For example, as shown in SEQ ID NO: 7, the DNA obtained from L428cell, a type of lymphoblastoid cell derived from a patient withHodgkin's disease, consists of the nucleotide sequence of SEQ ID NO: 1encoding the amino acid sequence of SEQ ID NO: 20, and anothernucleotide sequence encoding signal peptide which is linked to the5′-terminal in the nucleotide sequence of the SEQ ID NO: 1. Solublepolypeptides with the amino acid sequences of SEQ ID NOs: 22 to 25 areusually encoded by respective nucleotide sequences of SEQ ID NOs: 3 to6, which are usually used in a form where, as shown in the nucleotidesequences of SEQ ID NOs: 8 to 11, a nucleotide sequence encoding signalpeptide is linked to the 5′-terminal in the nucleotide sequences of SEQID NOs: 3 to 6. Such a DNA can be also obtained through usual chemicalsynthesis, and in any case, DNAs can be amplified to desired levels byPCR method once they become available. By the way, the amino acidsequences of SEQ ID NOs: 20 and 21 are described along with the aminoacid sequences for signal peptides in P. Parnet et al., The Journal ofBiological Chemistry, Vol. 271, pp. 3,967-3,970 (1996): This paperhowever makes neither suggestion nor teaching that the polypeptides withthe amino acid sequences of SEQ ID NOs: 20 and 21 do function as IL-18R.

[0035] Such DNA expresses the polypeptide when introduced into anappropriate host of microbe, animal or plant origin. The DNA of thisinvention is usually prepared into a recombinant DNA prior tointroduction into host. Such recombinant DNA, which consists of the DNAof this invention and an autonomously replicable vector, can be easilyprepared with usual recombinant DNA techniques, provided that the DNA isavailable. Examples of vectors which can receive the DNA of thisinvention are plasmid vectors including pKK223-3, pCDNAI/Amp, BCMGSNeo,pcDL-SRα, pKY4, pCDM8, pCEV4, pME18S and pEF-BOS. Autonomouslyreplicable vectors usually comprises other nucleotide sequences, forexample, promotor, enhancer, replication origin, terminator oftranscription, splicing sequence and/or selection marker whichfacilitate the expression of the DNA of this invention in particularhosts. Expression of the DNA becomes artificially regulatable uponexternal stimuli when it is used in combination with either heat shockprotein promotor or interferon a promotor as disclosed in JapanesePatent Kokai No. 163,368/95 by the same applicant.

[0036] Conventional methods are feasible in the insertion of the DNA ofthis invention into such vector. More particularly, a gene with the DNAof this invention and an autonomously replicable vector are firstdigested with restriction enzyme and/or ultrasonication, then theresultant DNA and vector fragments are ligated. Ligation of DNA andvector fragments become much easier when genes and vectors are digestedwith restriction enzymes specific to particular nucleotides, forexample, AccI, BamHI, BstXI, EcoRI, HindIII, NotI, PstI, SacI, SalI,SmaI, SpeI, XbaI and XhoI. To ligate DNA and vector fragments, they arefirst annealed, if necessary, then exposed to DNA ligase in vivo or invitro. The recombinant DNA thus obtained is unlimitedly replicable inhosts of microbe and animal origins. Such recombinant DNA is introducedinto an appropriate host, prior to use in preparation of thepolypeptide. Although conventional hosts of microbe, animal and plantorigins are feasible in this invention, it is preferable to choose ahost of yeast or mammalian origin in case that the final use of thepolypeptide is pharmaceuticals. Examples of host cells of mammalianorigin are epithelial cell, interstitial cell and hemopoietic cell ofhuman, monkey, mouse and hamster origins, in particular, 3T3 cell (ATCCCCL-92), C127I cell (ATCC CRL-1616), CHO-K1 cell (ATCC CCL-61), CV-1cell (ATCC CCL-70), COS-1 cell (ATCC CRL-1650), HeLa cell (ATCC CCL-2),MOP-8 cell (ATCC CRL-1709) and their mutant strains. To introduce theDNA of this invention into such a host, one can employ conventionalmethods, for example, DEAE-dextran method, calcium phosphatetransfection method, electroporation method, lipofection method,microinjection method and viral infection method using retrovirus,adenovirus, herpesvirus and vaccinia virus. To select among theresultant transformants a clone which is capable of producing thepolypeptide, the transformants are cultivated on culture medium,followed by selecting one or more clones where production of thepolypeptide is observed. Recombinant DNA techniques using host cells ofmammalian origin are detailed, for example, Jikken-Igaku-Bessatsu,Saibo-Kogaku Handbook (The handbook for the cell engineering), edited byToshio KUROKI, Masaru TANIGUCHI and Mitsuo OSHIMURA, published byYodosha. Co., Ltd., Tokyo, Japan (1992), and Jikken-Igaku-Bessatsu,Biomanual Series 3, Idenshi-Cloning-Jikken-Ho (The experimental methodsfor the gene cloning), edited by Takashi YOKOTA and Kenichi ARAI;published by Yodosha Co., Ltd., Tokyo, Japan (1993).

[0037] The transformant thus obtained produces and secretes thepolypeptide inside and/or outside the host cell when cultivated onculture medium. Such cultivation is feasible with conventional culturemedia directed to cultivation of transformants, which are usuallycomposed by adding to a bufferized water as base inorganic ions such assodium ion, potassium ion, calcium ion, phosphoric ion and chloric ion;minor elements, carbon sources, nitrogen sources, amino acids andvitamins which meet to the metabolism of particular hosts; and, ifnecessary, sera, hormones, cell growth factors and cell adhesionfactors. Particular media are, for example, 199 medium, DMEM medium,Ham's F12 medium, IMDM medium, MCDB 104 medium, MCDB 153 medium, MEMmedium, RD medium, RITC 80-7 medium, RPMI-1630 medium, RPMI-1640 mediumand WAJC 404 medium. One can obtain a culture product containing thepolypeptide by inoculating on such a culture medium a transformant in anamount of 1×10⁴-1×10⁷ cells/ml, preferably, 1×10⁵-1×10⁶ cells/ml, andsubjecting the transformant to suspension or monolayer culture at around37° C. for 1 day to 1 week, preferably, 2 to 4 days while replacing theculture medium with a fresh preparation, if necessary. The cultureproduct thus obtained usually contains about 1 μg/l to 1 mg/lpolypeptide, dependently of the type of transformant and cultivationconditions.

[0038] The culture product obtained in this. way is first subjected toultrasonication, cell-lytic enzyme and/or detergent to disrupt cells, ifnecessary, then polypeptides are separated from the cells or cell debrisby filtration and centrifugation, followed by purification. In thepurification, a culture product which has been separated from cell orcell debris is subjected to conventional methods common in purificationof biologically-active proteins, for example, salting-out, dialysis,filtration, concentration, fractional precipitation, ion-exchangechromatography, gel filtration chromatography, adsorptionchromatography, isoelectric focusing chromatography, hydrophobicchromatography, reversed phase chromatography, affinity chromatography,gel electrophoresis and isoelectric focusing gel electrophoresis whichare used in combination, if necessary. The purified polypeptide is thenconcentrated and lyophilized into liquid or solid to meet to its finaluse. The IL-18 and monoclonal antibody, disclosed in Japanese PatentKokai No. 193,098/96 and Japanese Patent Application No. 356,426/96 bythe same applicant, are very useful in purification of the polypeptide:Immunoaffinity chromatographies using these do yield a high-puritypreparation of the polypeptide with minimized costs and labors.

[0039] The polypeptide of this invention exhibits a remarkable efficacyin treatment and prevention of various diseases resulting from excessiveimmunoreaction because in mammals including human, the polypeptiderecognizes and binds IL-18 which may activate immune system. Immunesystem, which is in nature to defend living bodies from harmful foreignsubstances, may cause unfavorable results in living bodies because ofits nature. When mammals receive a graft of organ, for example, skin,kidney, liver, heart and bone marrow, the rejection reaction andimmunoreaction against alloantigen may activate T-cells, resulting inthe occurrence of inflammation and proliferation of lymphocytes. Similarphenomena are observed in case that host receives the invasion byheteroantigens, for example, allergens, which are not recognized asself. In autoimmune diseases, allergic reactions are induced bysubstances which must be recognized as self. The polypeptide of thisinvention exhibits a remarkable efficacy in treatment and prevention ofvarious diseases resulting from such an immunoreaction because thepolypeptide suppresses or regulates the immunoreaction when administeredin mammals including human. Thus, the wording “susceptive diseases” asreferred to in this invention shall mean all the diseases resulting fromaugmented immunoreaction which can be treated and/or prevented by thedirect or indirect action of IL-18R: Particular susceptive diseases are,for example, rejection reactions associated with a graft of organ asdescribed above, autoimmune and allergic diseases including perniciousanemia, atrophic gastritis, insulin-resistant diabetes, Wegenergranulomatosis, discoid lupus erythematosus, ulcerative colitis, coldagglutinin-relating diseases, Goodpasture's syndrome, primary biliarycirrhosis, sympathetic ophtalmitis, hyperthyroidism, juvenile onset typediabetes, Sjögren syndrome, autoimmune hepatitis, autoimmune hemolyticanemia, myasthenia gravis, systemic scleroderma, systemic lupuserythematosus, polyleptic cold hemoglobinuria, polymyositis,periarteritis nodosa, multiple sclerosis, Addison's disease, purpurahemorrhagica, Basedow's disease, leukopenia, Behcet's disease,climacterium praecox, rheumatoid arthritis, rheumatopyra, chronicthyroiditis, Hodgkin's disease, HIV-infections, asthma, atopicdermatitis, allergic nasitis, pollinosis and apitoxin-allergy. Inaddition, the polypeptide of this invention is efficacious in treatmentand prevention of septic shock which results from production oradministration of excessive IFN-γ.

[0040] Thus, the agent for susceptive disease, which contains aseffective ingredient the polypeptides of this invention, would find avariety of uses as anti-autoimmune-diseases, anti-allergies,anti-inflammatories, immunosuppressants, hematopoietics, leukopoietics,thrombopoietics, analgesics and antipyretics directed to treatmentand/or prevention of susceptive diseases as illustrated in the above.The agent according to this invention is usually prepared into liquid,suspension, paste and solid forms which contain the polypeptide in anamount of 0.00001-100 w/w %, preferably, 0.0001-20 w/w %, dependently onthe forms of agents as well as on the types and symptoms of susceptivedisease.

[0041] The agent for susceptive diseases according to this inventionincludes those which are solely composed of the polypeptide, as well asincluding those in composition with, for example, one or morephysiologically-acceptable carriers, excipients, diluents, adjuvants,stabilizers and, if necessary, other biologically-active substances:Examples of such stabilizer are proteins such as serum albumins andgelatin; saccharides such as glucose, sucrose, lactose, maltose,trehalose, sorbitol, maltitol, mannitol and lactitol; and buffers whichare mainly composed of phosphate or succinate. Examples of thebiologically-active substances usable in combination are FK506,glucocorticoid, cyclophosphamide, nitrogen mustard,triethylenethiophosphoramide, busulfan, pheniramine mustard,chlorambucil, azathioprine, 6-mercaptopurine, 6-thioguanine,6-azaguanine, 8-azaguanine, 5-fluorouracil, cytarabine, methotrexate,aminopterin, mitomycin C, daunorubicin hydrochloride, actinomycin D,chromomycin A₃, bleomycin hydrochloride, doxorubicin hydrochloride,cyclosporin A, L-asparaginase, vincristine, vinblastine, hydroxyurea,procarbazine hydrochloride, adrenocortical hormone and auri colloid;receptor antagonists to cytokines other than IL-18, for example,antibodies respectively against interleukin-1 receptor protein,interleukin-2 receptor protein, interleukin-5 receptor protein,interleukin-6 receptor protein, interleukin-8 receptor protein andinterleukin-12 receptor protein; and antagonists respectively againstTNF-α receptor, TNF-β receptor, interleukin-1 receptor, interleukin-5receptor and interleukin-8 receptor.

[0042] The agent for susceptive diseases according to this inventionincludes pharmaceuticals in minimal dose unit: The wording“pharmaceutical in minimal dose unit” represents those which areprepared into a physically united form suitable for prescription andalso allowed to contain the polypeptide in an amount corresponding toits single dose or multiple (up to 4-fold) or divisor (up to {fraction(1/40)}) thereof: Examples of such form are injection, liquid, powder,granule, tablet, capsule, sublingual, ophthalmic solution, nasal dropand suppository. The agent for susceptive diseases according to thisinvention can be administrated through both oral and parenteral routesto exhibit in each case a remarkable efficacy in treatment andprevention of susceptive diseases. More particularly, the polypeptide isadministered through oral route or parenteral route such as intradermal,subcutaneous, intramuscular or intravenous route at a dose of about 1μg/time/adult to about 1 g/time/adult, preferably, about 10μg/time/adult to about 100 mg/time/adult 1 to 4 times/day or 1 to 5times/week over 1 day to 1 year.

[0043] The DNA which encodes the polypeptide of this invention is usefulin “gene therapies”. Particularly, in usual gene therapies, the DNA ofthis invention is first inserted in a vector derived from virus such asretrovirus, adenovirus or adeno-associated virus and, alternatively,embedded in either cationic- or membrane fusible-liposomes, then theinserted or embedded DNA is directly injected in a patient with an IL-18susceptive disease and, alternatively, introduced into lymphocytes,which have been collected from the patient, and implanted in thepatient. In adoptive immuno gene therapies, by introducing the DNA ofthis invention into effector cells similarly as in the usual genetherapies, the cytotoxicity of effector cells against tumors andvirus-infected cells is enhanced and this would strengthen adoptiveimmunotherapy. In tumor vaccine gene therapy, tumor cells, which havebeen extracted from a patient, are introduced with the DNA of thisinvention similarly as in the usual gene therapies, allowed toproliferate in vitro to a prescribed level and then self-transplanted tothe patient: The transplanted tumor cells act as vaccine in the body ofthe patient, exhibiting a strong and antigen-specific antitumorimmunity. Thus, the DNA of this invention exhibits a remarkable efficacyin gene therapies for various diseases including, for example, malignanttumors, vial diseases, infections and autoimmune diseases, as well as insuppression of rejection reaction and excessive immunoreactionassociated with grafts of organs and allergic diseases. Generalprocedures for gene therapies are detailed in Jikken-Igaku-Bessatsu,Biomanual UP Series, Idenshichiryo-no-Kisogijutsu (Basic techniques forthe gene therapy), edited by Takashi SHIMADA, Izumi SAITO, and KeiyaOZAWA, published by Yodosha Co., Ltd., Tokyo, Japan (1996).

[0044] Further, the polypeptide of this invention is useful in affinitychromatography and labelled assay directed to purification and detectionof IL-18 because the polypeptide bears properties of recognizing andbinding IL-18. In addition, the polypeptide of this invention, inparticular, that in soluble form is useful in screening in vivo or invitro agonists and antagonists to IL-18. Furthermore, the agent toneutralize IL-18 containing as effective ingredient the polypeptide andthe method to neutralize IL-18 where IL-18 is exposed to the polypeptideare useful in treatment of various diseases which result from productionand administration of excessive IL-18.

[0045] The following Examples are to illustrate the way of practicingthis invention. The techniques employed in Examples 1 to 3 are common inthis field as detailed, for example, Jikken-Igaku-Bessatsu, Saibo-KogakuHandbook (The handbook for the cell engineering), edited by ToshioKUROKI, Masaru TANIGUCHI and Mitsuo OSHIMURA, published by Yodosha. Co.,Ltd., Tokyo, Japan (1992), and Jikken-Igaku-Bessatsu, Biomanual Series3, Idenshi-Cloning-Jikken-Ho (The experimental methods for the genecloning), edited by Takashi YOKOTA and Kenichi ARAI, published byYodosha Co., Ltd., Tokyo, Japan (1993).

EXAMPLE 1 Preparation and Characterization of IL-18R Example 1-1Preparation of IL-18R

[0046] Newborn hamsters were intraperitoneally injected with ananti-lymphocyte antibody of rabbit origin to suppress their possibleimmunoreaction, subcutaneously injected at their dorsal areas with about5×10⁵ cell/animal of L428 cells (FERM BP-5777), a type of lymphoblastoidcell derived from a patient with Hodgkin's disease, and fed in usualmanner for 3 weeks. The tumor masses, subcutaneously occurred, about 10g each, were extracted, disaggregated and washed in usual manner inserum-free RPMI-1640 medium (pH 7.4), thus obtaining proliferated cells.

[0047] The proliferated cells were added with a mixture solution (volumeratio of 9:1) of 0.83 w/v % NH₄Cl and 170 mM Tris-HCl buffer (pH 7.7) inan amount 10-fold larger than the wet weight of the cells, stirred andcollected by centrifugation at 2,000 rpm for 10 minutes. The cells werethen suspended in an appropriate amount of phosphate buffered saline(hereinafter abbreviated as “PBS”), stirred, collected by centrifugationat 2,000 rpm, resuspended to give a cell density of about 1×10⁸ cells/mlin 10 mM Tris-HCl buffer (pH 7.2) with 1 mM MgCl₂ and disrupted with“POLYTRON”, a cell disrupter commercialized by Kinematica AG,Littau/Lucerne, Switzerland. The resultant was added with 10 mM Tris-HClbuffer (pH 7.2) containing both 1 mM MgCl₂ and 1M sucrose to give afinal sucrose concentration of 0.2M, and centrifuged at 1,000 rpm tocollect the supernatant which was then centrifuged at 25,000 rpm for 60minutes, followed by collecting the precipitate. The precipitate wasadded with adequate amounts of 12 mM3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid(hereinafter abbreviated as “CHAPS”), 10 mM ethylenediaminetetraacetaticacid (hereinafter abbreviated as “EDTA”) and 1 mMphenylmethylsulfonylfluoride, stirred at 4° C. for 16 hours, andcentrifuged at 25,000 rpm for 60 min, followed by collecting thesupernatant.

[0048] The supernatant was charged to a column of “WHEAT GERM LECTINSEPHAROSE 6B”, a gel product for affinity chromatography commercializedby Pharmacia LKB Biotechnology AB, Uppsala, Sweden, pre-equilibrated inPBS with 12 mM CHAPS, and the column was washed with PBS containing 12mM CHAPS, and then charged with PBS containing both 0.5 MN-acetyl-D-glucosamine and 12 mM CHAPS while monitoring the proteincontent in the eluate with the absorbance of ultraviolet at a wavelength of 280 nm. The fractions with an absorbance of 0.16-0.20 werecollected and pooled, thus obtaining about 25 liters of aqueous solutionwith a protein content of about 1 mg/ml per 10¹² starting cells.

[0049] A small portion of the solution was sampled, added with 4 nghuman IL-18which had been ¹²⁵I-labelled in usual manner, incubated at 4°C. for 1 hour, added with appropriate amounts of “POLYETHYLENE GLYCOL6000”, polyethylene glycol preparation with an averaged molecular weightof 6,000 daltons, commercialized by E. Merck, Postfach, Germany, andallowed to stand under ice-chilling conditions for 30 minutes to effectbinding reaction. The reaction product was centrifuged at 6,000 rpm for5 minutes and the resultant precipitate was collected to determine thelevel of radioactivity. In parallel, there was provided another sectionsas control in which 3 μg non-labelled human IL-18 was used along with¹²⁵I-labelled human IL-18 and treated similarly as above. Comparisonwith control revealed that the radioactivity of the precipitate from thesample solution was significantly higher. This indicated that theaqueous solution obtained in the above did contain IL-18R and the I-18Rrecognized and bound IL-18 when exposed to IL-18.

Example 1-2 Binding Ability to Monoclonal Antibody

[0050] L428 cells (FERM BP-5777) were suspended in RPMI-1640 medium (pH7.4), supplemented with 0.1 v/v % bovine serum albumin and alsocontaining 0.1 v/v % NaN₃, to give a cell density of 4×10⁷ cells/ml,while monoclonal antibody MAb#117-10C specific to human IL-18R, obtainedby the method described in Japanese Patent Application No. 356,426/96 bythe same applicant, was dissolved in another preparation of RPMI-1640medium supplemented with 0.1 w/v % bovine serum albumin to givedifferent concentrations of 0.019 μg/ml, 0.209 μg/ml, 2.3 μg/ml, 25.3μg/ml and 139.5 μg/ml.

[0051] Fifty microliter aliquots of the cell suspension prepared in theabove were mixed with 50 μl of either solution with different monoclonalantibody concentrations, agitated at 4° C. for 2 hours, added with 50 μlof RPMI-1640 medium supplemented with 0.1 v/v % bovine serum albumin andalso containing 4 ng ¹²⁵I-labelled human IL-18 prepared in usual manner,and agitated at the same temperature for an additional 30 minutes.Subsequently, each cell suspension was added with 200 μl mixturesolution (volume ratio 1:1) of dibutylphthalate and diocthylphtalate andcentrifuged at 10,000 rpm and 20° C. for 5 minutes, followed bycollecting the resultant precipitates containing the cells which werethen determined for radioactivity using “MODEL ARC-300”, a gamma-raycounter commercialized by Aloka Co., Ltd, Tokyo, Japan.

[0052] In parallel, there were provided additional two sections wherethe monoclonal antibody was neglected, while 4 ng ¹²⁵I-labelled humanIL-18 was treated similarly as in the sample testing section with orwithout 4 micrograms of non-labelled human IL-18 (hereinafter referredto as “non-specific binding section” and “whole binding section”respectively). The levels of radioactivity found in “non-specificbinding section” and “whole binding section” were put in Formula 1together with that found in the sample testing section to calculatepercent inhibition. The results were as shown in FIG. 1.

Formula 1

[0053]${{Percent}\quad {Inhibition}} = {\frac{\left( {{Whole}\quad {binding}} \right) - ({Testing})}{\left( {{Whole}\quad {binding}} \right) - \left( {{Non}\text{-}{specific}\quad {binding}} \right)} \times 100}$

[0054] Fifty microliter aliquots of an IL-18R in aqueous solutionobtained by the method in Example 1-1 were added with 50 μl solutionwith different concentrations for monoclonal antibody MAb #117-10Cprepared similarly as above, agitated at 4° C. for 2 hours, added with 4ng ¹²⁵I-labelled human IL-18, and agitated at 4° C. for an. additional30 minutes. Subsequently, each mixture was added with 50 μl of 4 mg/mlγ-globulin, allowed to stand under ice-chilling conditions for 30minutes, added with 250 μl of PBS with 20 w/v % polyethylene glycol,allowed to stand under ice-chilling conditions for an additional 30minutes, and centrifuged at 6,000 rpm at 4° C. for 5 minutes, followedby collecting the resultant precipitates which were then determined forradioactivity similarly as above.

[0055] At the same time, there were provided additional two sectionswhere the monoclonal antibody was neglected, while 4 ng of ¹²⁵I-labelledhuman IL-18 were treated similarly as in the sample testing section withor without 4 μg of non-labelled human IL-18 (hereinafter referred to as“whole binding section” and “non-specific binding section”respectively). The levels of radioactivity found in these two sectionwere put in Formula 1 together in that found in the sample testingsection to calculate percent inhibition. The results were as shown inFIG. 1.

[0056] As seen in FIG. 1, in both cases of using L428 cell and IL-18R insolution, the binding of IL-18 to L428 cell and IL-18R were inhibitedmuch more as the concentration of monoclonal antibody MAb #117-10Celevated. This indicated that the monoclonal antibody MAb #117-10C wasbound to the possible IL-18R on the surface of L428 cell in a fashioncompeting with IL-18, as well as that the aqueous solution obtained bythe method in Example 1 did contain a protein capable of recognizingIL-18 or IL-18R and the monoclonal antibody MAb #117-10C specificallyreacted with the IL-18R.

Example 1-3 Western Blotting

[0057] A portion of the IL-18R in aqueous solution obtained by themethod in Example 1 was sampled, added with ⅔ volume of a mixturesolution of 2.5 w/v % sodium dodecyl sulfate and 50 v/v % glycerol,incubated at 37° C. for 1 hour, and separated into respectiveproteinaceous components on conventional SDS-PAGE using 10-20% gradientgel but using no reducing agent. The proteinaceous components on the gelwere transferred in usual manner to a nitrocellulose membrane which wasthen soaked for 1 hour in an appropriate amount of 50 mM Tris-HCl buffer(pH 7.5) with 10 μg/ml of monoclonal antibody MAb #117-10C obtained bythe methods described in Japanese Patent Application No. 356,426/96 bythe same applicant, 10 v/v % “BLOCK ACE”, an immobilizing agentcommercialized by Dainippon Seiyaku Co., Ltd., Osaka, Japan, and 0.05v/v % “TWEEN 20”, a detergent commercialized by City Chemical Corp., NewYork, U.S.A., and washed in 50 mM Tris-HCl buffer (pH 7.5) with 0.05 v/v% Tween 20 to remove the remaining antibody. The membrane was thensoaked in Tris-HCl buffer (pH 7.5) with an appropriate amount of ananti-mouse immunoglobulin antibody of rabbit origin prelabelled withhorse radish peroxidase, 10 v/v % “BLOCK ACE” and 0.05 v/v % “TWEEN 20”for 1 hour to effect reaction, washed in 50 mM Tris-HCl buffer (pH 7.5)with 0.05 v/v % “TWEEN 20” and developed using “ECL kit”, a kit fordevelopment commercialized by Amersham Corp., Arlington Heights, U.S.A.

[0058] At the same time, there was provided another section without themonoclonal antibody MAb #117-10C as control and it was treated similarlyas above. The molecular weight markers were bovine serum albumin (67,000daltons), ovalbumin (45,000 daltons), carbonic anhydrase (30,000daltons), trypsin inhibitor (20,100 daltons) and α-lactoalbumin (14,000daltons). The results were as shown in FIG. 2.

[0059] In the gel electrophoresis in FIG. 2, Lane 2 (with monoclonalantibody) bore a distinct band of IL-18R which was never found in Lane 3(without monoclonal antibody).

Example 1-4 Inhibition of IL-18 Activity

[0060] KG-1 cells (ATCC CCL246), an established cell line derived from apatient with acute myelogenous leukemia, were suspended in RPMI-1640medium (pH 7.2), supplemented with 10 v/v % fetal bovine serum and alsocontaining 100 μg/ml kanamycin and 18.8 mM Na₂HPO₄, to give a celldensity of 1×10⁷ cells/ml, added with monoclonal antibody MAb #117-10C,obtained by the method described in Japanese Patent Application No.356,426/96 by the same applicant, to give a concentration of 10 μg/mland incubated at 37° C. for 30 minutes.

[0061] The KG-1 cells in suspension were distributed on 96-wellmicroplate to give respective amounts of 50 μl/well, added with 50 μl ofhuman IL-18 which had been dissolved in a fresh preparation of the samemedium to give respective concentrations of 0 ng/ml, 1.56 ng/ml, 3.12ng/ml, 6.25 ng/ml, 12.5 ng/ml and 25 ng/ml, further added with 50μl/well of 5 μg/ml lipopolysaccharide in a fresh preparation of theabove medium, and incubated at 37° C. for 24 hours, after which eachsupernatant was collected and determined for IFN-γ content byconventional enzyme immunoassay. In parallel, there were providedadditional sections without the monoclonal antibody MAb #117-10C forrespective IL-18 concentrations as control and they were treatedsimilarly as above. The results were as shown in FIG. 3. The IFN-γcontents in FIG. 3 were calibrated with reference to the standardizedIFN-γ preparation Gg23-901-530 available from the InternationalInstitute of Health, USA, and expressed in the International Unit (IU).

[0062] The results in FIG. 3 indicated that the presence of monoclonalantibody MAb #117-10C inhibited the induction of IFN-γ by IL-18 in KG-1cell as immunocompetent cell. This also indicated that monoclonalantibody MAb #117-10C blocked the IL-18R on the surface of KG-1 cell ina fashion competing with IL-18, thus preventing the signal transductionof IL-18 to KG-1 cell.

Example 1-5 Purification of IL-18R

[0063] Seventy-eight milligrams of a monoclonal antibody MAb #117-10C,obtained by the method described in Japanese Patent Application No.356,426/96 by the same applicant, was dissolved in an appropriate amountof distilled water and the solution was dialyzed against borate buffer(pH 8.5) with 0.5M NaCl at 4° C. for 16 hours. Thereafter, in usualmanner, an appropriate amount of “CNBr-ACTIVATED SEPHAROSE 4B”, aCNBr-activated gel, commercialized by Pharmacia LKB Biotechnology AB,Uppsala, Sweden, was added to the dialyzed solution and allowed to reactat 4° C. for 18 hours under gentle stirring conditions to immobilize themonoclonal antibody MAb #117-10C on the gel.

[0064] The gel was packed into column in a plastic cylinder,equilibrated with 2 mM CHAPS, charged with an IL-18R in aqueous solutionobtained by the method in Example 1-1, and applied with PBS with 12 mMCHAPS to remove non-adsorbed components. The column was then appliedwith 35 mM ethylamine containing 2 mM CHAPS (pH 10.8) while collectingthe eluate in every 8 ml fractions which were then checked for presenceof IL-18R by the method in Example 1-1 using ¹²⁵I-labelled human IL-18.The chromatogram obtained in this operation was as shown in FIG. 4.

[0065] As seen in FIG. 4, IL-18R was eluted in a single sharp peak whenimmunoaffinity chromatography using monoclonal antibody MAb #117-10C wasapplied to a mixture of IL-18R and contaminants such as the aqueoussolution of IL-18R in Example 1-1. The fractions corresponding to thissingle peak were collected, pooled and lyophilized, thus obtaining apurified IL-18R in solid form.

[0066] Thereafter, a portion of the purified IL-18R was sampled,incubated in PBS at 100° C. for 5 minutes, and determined for residualactivity by the method in Example 1-2, resulting in no binding to IL-18which proved that IL-18R was inactivated by heating. This would supportthat the nature of this receptor is proteinaceous.

[0067] Further, a portion of the purified IL-18R obtained in the abovewas dissolved in an appropriate amount of PBS, dialyzed against PBS atambient temperature overnight, added with an appropriate amount of¹²⁵I-labelled human IL-18 prepared by the method in Example 1-1 and 1 mM“BS³”, a polymerizing agent commercialized by Pierce, Rockford, U.S.A.,and allowed to stand at 0° C. for 2 hours to form a conjugate of IL-18Rand ¹²⁵I-labelled human IL-18. The reaction mixture was added withTris-HCl buffer (pH 7.5), allowed to stand at 0° C. for an additional 1hour to suspend the conjugation reaction, separated into respectiveproteinaceous components on SDS-PAGE using a set of molecular weightmarkers and dithiothreitol as reducing agent, and subjected toautoradiogram analysis.

[0068] The apparent molecular weight for this conjugate of IL-18R and¹²⁵I-labelled human IL-18 was about 50,000 to 200,000 daltons whenestimated with reference to the mobility of molecular weight markers onthe autoradiogram. Since the molecular weight of IL-18 is about 20,000daltons, the molecular weight of IL-18R can be estimated about30,000-180,000 daltons on the assumption that IL-18R binds one humanIL-18 molecule.

Example 1-6 Peptide Mapping of IL-18R

[0069] A purified IL-18R obtained by the method in Example 1-5 waselectrophoresed on SDS-PAGE using 7.5 w/v % gel with 2 w/v %dithiothreitol as reducing agent, and the gel was then soaked for 5minutes in a mixture solution of 40 v/v % aqueous methanol and 1 v/v %acetic acid with 0.1 w/v % Coomassie Brilliant Blue for development, andsoaked for an additional 2 hours for destaining in the same solution butwithout Coomassie Brilliant Blue, after which the stained part in thegel, molecular weight of 80,000-110,000 daltons, was cut off, added with50 v/v % aqueous acetonitrile containing 0.2 M (NH₄)₂CO₃ and repeatedlyagitated at ambient temperature. Thereafter, the gel slices werelyophilized, added with 0.2M (NH₄)₂CO₃ (pH 8.0), allowed to stand for 5minutes to effect swelling, added with appropriate amounts of 1 mMhydrochloric acid with 0.1 μg/μl “SEQUENCING GRADE MODIFIED TRYPSIN”, areagent of trypsin commercialized by Promega Corp., Madison, U.S.A., and0.2 M (NH₄)₂CO₃ (pH 8.9), and allowed to react at 37° C. overnight.After suspending with 10 v/v % aqueous acetic acid solution, thereaction mixture was added with a mixture solution of 0.1 v/v %trifluoroacetic acid and 60 v/v % aqueous acetonitrile and agitated atambient temperature, after which the resultant supernatant wascollected, concentrated in vacuo and centrifugally filtered, thusobtaining a concentrate with peptide fragments.

[0070] The concentrate was charged to “μRPC C2/C18-SC2.1/10”, a columnfor high-performance liquid chromatography commercialized by PharmaciaLKB Biotechnology AB, Uppsala, Sweden, pre-equilibrated with 0.065 v/v %trifluoroacetic acid, and then applied at a flow rate of 100 μl/min with0.055 v/v % trifluoroacetic acid containing 80 v/v % aqueousacetonitrile under liner gradient of acetonitrile increasing from 0 to80 v/v over 160 minutes immediately after application of the eluent.While monitoring the absorbance at a wavelength of 240 nm, the eluatewas fractioned to separately collect respective peptide fragments whicheluted about 45, 50, 55, 58, 62, 72, 75 and 77 minutes after applicationof the eluent. The peptide fragments (hereinafter referred to as“peptide fragment 1”, “peptide fragment 2”, “peptide fragment 3”,“peptide fragment 4”, “peptide fragment 5”, “peptide fragment 6”,“peptide fragment 7” and “peptide fragment 8” in the order of elution)were analyzed in usual manner for amino acid sequence using “MODEL473A”, a protein sequencer commercialized by Perkin-Elmer Corp.,Norwalk, U.S.A, revealing that the peptide fragments 1 to 8 bore theamino acid sequences of SEQ ID NOs: 12 to 19 respectively. The peptidemap obtained-by this operation was as shown in FIG. 5.

EXAMPLE 2 Preparation of DNA Example 2-1 Preparation of Total RNA

[0071] In usual manner, L428 cells (FERM BP-5777) were suspended inRPMI-1640 medium (pH 7.2) supplemented with 10 v/v % fetal bovine serum,and proliferated at 37° C. while scaling up the cultivation. When thecell density reached a prescribed level, the proliferated cells werecollected, suspended in 10 mM sodium citrate (pH 7.0) containing both 6Mguanidine isothiocyanate and 0.5 w/v % sodium N-laurylsarcosinate, andthen disrupted with a homogenizer.

[0072] Aliquots of 0.1M EDTA (pH 7.5) containing 5.7M CsCl₂ were placedin 35 ml-reaction tubes, poured with the cell disruptant obtained in theabove in layer over the EDTA in each tube, and subjected toultracentrifugation at 20° C. at 25,000 rpm for 20 hours to collect theRNA fraction. The RNA fraction was distributed in 15 ml-centrifugationtubes, added with an equivolume each of a mixture solution ofchloroform/1-butanol (volume ratio 4:1), agitated for 5 minutes andcentrifuged at 4° C. at 10,000 rpm for 10 minutes, after which theaqueous layer was collected, added with 2.5-fold volume of ethanol andallowed to stand at −20° C. for 2 hours to precipitate the total RNA.The precipitate was collected, washed with 75 v/v % aqueous ethanol, andthen dissolved in 0.5 ml of sterilized distilled water to obtain asolution of the total RNA originating from L428 cell.

Example 2-2 Preparation of mRNA

[0073] An aqueous solution containing total RNA solution obtained by themethod in Example 2-1 was added with 0.5 ml of 10 mM Tris-HCl buffer (pH7.5), containing both 1 mM EDTA and 0.1 w/v % sodiumN-laurylsarcosinate, to bring the total volume to 1 ml. The mixturesolution was added with 1 ml of “OLIGOTEX™-dT30 <SUPER>”, a latex withan oligonucleotide of (dT)₃₀ commercialized by Japan Roche K. K., Tokyo,Japan, reacted at 65° C. for 5 minutes and rapidly cooled in anice-chlling bath. Thereafter, the reaction mixture was added with 0.2 mlof 5 mM NaCl, incubated at 37° C. for 10 minutes, centrifuged at 10,000rpm for 10 minutes to collect the resultant precipitate in pellet formwhich was then suspended in 0.5 ml of sterilized distilled water andincubated at 65° C. for 5 minutes to desorb the mRNA from the latex. Theobtained solution was added with an appropriate amount of ethanol, andthe resultant precipitate was collected and lyophilized to obtain asolid of mRNA.

Example 2-3 Preparation of DNA Fragment Encoding Polypeptide

[0074] Four microliters of 25 mM MgCl₂, 2 μl of 100 mM Tris-HCl buffer(pH 8.3) containing 500 mM KCl, 1 μl of 25 mM dNTP mix, 0.5 μl of 40units/μl ribonuclease inhibitor and 1 μl of 200 units/μl reversetranscriptase were placed in a 0.5 ml-reaction tube, added with 10 ng ofan mRNA, obtained by the method in Example 2-2, along with anappropriate amount of random hexanucleotides, and added with sterilizeddistilled water to bring the total volume of 20 μl. The obtained mixturewas incubated first at 42° C. for 20 minutes, then at 99° C. for 5minutes to suspend the reaction, thus obtaining a reaction mixturecontaining a first strand cDNA.

[0075] Twenty microliters of the reaction mixture was added with 1 μl of2.5 units/μl “CLONED Pfu POLYMERASE”, a DNA polymerase commercialized byStratagene Cloning Systems, California, U.S.A., 10 μl of the reactionbuffer and 1 μl of 25 mM dNTP mix, both commercialized by StratageneCloning Systems, added with 0.1 μg each of oligonucleotides as sense andantisense primers having respective nucleotide sequences as shown with5′-TCAGTCGACGCCACCATGAATTGTAGAGAA-3′ and5′-GAAGCGGCCGCATCATTAAGACTCGGAAAGAAC-3′ which had been prepared on thebasis of the amino acid sequence described in P. Parnet et al., TheJournal of Biological Chemistry, Vol. 271, pp. 3967-3970 (1996), addedwith sterile distilled water to bring the total volume to 100 μl. Theresultant mixture was subjected first to 3-time cycles of incubating at95° C. for 1 minute, 42° C. for 2 minutes and 72° C. for 3 minutes inthe given order, then to 35-time cycles of incubating at 95° C. for 1minute, 60° C. for 2 minutes and 72° C. for 3 minutes in the given orderto effect PCR reaction.

[0076] Fifty nanograms of the obtained PCR product was added with 1 ngof “pCR-Script Cam SK(+)”, a plasmid vector commercialized by StratageneCloning Systems, California, U.S.A., and then subjected to ligationreaction at 16° C. for 2 hours using “DNA LIGATION KIT VERSION 2”, a DNAligation kit commercialized by Takara Syuzo, Co., Ltd., Otsu, Shiga,Japan, to insert the DNA fragment of the PCR product in the plasmidvector. A portion of the reaction product was sampled and used in usualmanner to transform “XL1-BLUE MRF′ KAN”, an Escherichia coli straincommercialized by Stratagene Cloning Systems, California, U.S.A.

EXAMPLE 3 Preparation of Recombinant DNA

[0077] A transformant obtained by the method in Example 2-3 wasinoculated in LB medium containing 30 μg/ml chloramphenicol andcultivated at 37° C. for 18 hours, after which the cells were collectedfrom the culture and treated in usual manner to obtain the plasmid DNA.After confirming by the dideoxy method that the plasmid DNA containedthe nucleotide sequence of SEQ ID NO: 7, the plasmid DNA was exposed toboth restriction enzymes NotI and SalI, and 100 ng of the obtained DNAfragment was added with 10 ng of “pcDNAI/Amp”, a plasmid vector with amodified multiple cloning site, commercialized by InvitrogenCorporation, San Diego, U.S.A., which had been predigested with bothrestriction enzymes NotI and XhoI, and subjected to ligation reaction at16° C. for 2 hours using “LIGATION KIT VERSION 2”, a ligation kitcommercialized by Takara Syuzo Co., Ltd., Otsu, Shiga, Japan. A portionof the reaction product was sampled and introduced in usual manner into“XL1-BLUE MRF′ KAN”, a strain of Escherichia coli commercialized byStratagene Cloning Systems, California, U.S.A., to obtain a transformant“cDNA/HuIL-18R” which contained a recombinant DNA “pcDNA/HuIL-18R” ofthis invention. The recombinant DNA “pcDNA/HuIL-18R” was analyzed inusual manner, revealing that in the recombinant DNA, a DNA “IL-18RcDNA”, which contained the nucleotide sequence of SEQ ID NO: 1 encodingthe polypeptide of this invention, was linked downstream the cytomegalovirus promotor Pcmv, as shown in FIG. 6.

EXAMPLE 4 Preparation of Transformant

[0078] A transformant “cDNA/HuIL-18R” obtained by the method in Example3 was inoculated in LB medium (pH 7.5) containing 100 μg/ml ampicillinand cultured at 37° C. for 18 hours, after which the cells werecollected from the culture and treated in usual manner to obtain theplasmid DNA. Separately, COS-1 cell (ATCC CRL-1650), a fibroblastic cellline derived from a kidney of African green monkey was proliferated inusual manner, and 20 micrograms of the plasmid DNA obtained in the abovewas introduced by conventional electroporation method into 1×10⁷ COS-1cells to obtain transformant cells which contained the DNA of thisinvention.

EXAMPLE 5 Preparation of Polypeptide

[0079] DMEM medium (pH 7.2) supplemented with 10 v/v % fetal bovineserum was distributed in flat-bottomed culture bottles, inoculated withtransformant cells, obtained by the method in Example 4, to give a celldensity of 1×10⁵ cells/ml, and cultured at 37° C. in 5 v/v % CO₂incubator for 3 days. After removing the supernatant from the culture,PBS containing both 5 mM EDTA and 0.02 w/v % NaN₃ was placed in theculture bottles to desorb the proliferated cells.

[0080] After washing in PBS, the proliferated cells were rinsed in abuffer containing 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl₂ and 0.1 mM EDTA(hereinafter referred to as “hypotonic buffer”), and suspended in afresh preparation of the hypotonic buffer to give a cell density of2×10⁷ cells/ml. The cell suspension was homogenized with a Dounce-typehomogenizer under ice-chilling conditions, and the resultant homogenatewas centrifuged at 15,000 rpm at 5 minutes to remove both cell nucleiand intact cells, and dialyzed overnight against PBS containing 2 mMCHAPS.

[0081] The dialyzed product was charged to a column of immobilizedmonoclonal antibody MAb #117-10C, prepared by the method in Example 1-5,which was then applied with PBS containing 12 mM CHAPS to removenon-adsorbed components. Thereafter, the column was applied with 35 mMethylamine (pH 10.8) containing 2 mM CHAPS while collecting andfractionating the eluate, was applied to the column, and the eluate wasfractionally collected. Each fraction was then checked for presence ofthe polypeptide of human origin by the method in Example 1-1 using¹²⁵I-labelled human IL-18, selected and pooled to obtain per 10⁸starting cells about 2 ml of an aqueous solution which contained apolypeptide with the amino acid sequence of SEQ ID NO: 20. The proteincontent in the solution was about 10 μg/ml.

[0082] The polypeptide thus obtained was studied for physicochemicalproperties by the methods in Example 1. As the result, the polypeptideobtained in this Example contained each amino acid sequence in SEQ IDNOs: 12 to 19 as partial amino acid sequences, as well as exhibitingphysiological activities which were similar to those of the IL-18R fromL428 cell.

EXAMPLE 6 Soluble Polypeptide from Human Origin Example 6-1 Preparationof Recombinant DNA

[0083] One nanogram of a recombinant DNA “pcDNA/HuIL-18R” obtained bythe method in Example 3, 10 μl of 10×PCR buffer and 1 μl of 25 mM dNTPmix were placed in 0.5 ml-reaction tube, added with 1 microliter of 2.units/microliter Pfu DNA polymerase, added with appropriate amounts ofoligonucleotides as sense and antisense primers having respectivenucleotide sequences as shown with5′-TCAGTCGACGCCACCATGAATTGTAGAGAATTA-3′ and5′-GAAGCGGCCGCATCATTATCTTGTGAAGACGTG-3′, and with sterile distilledwater to bring the total volume to 100 μl. The resultant mixture wassubjected first to 3-time cycles of incubating at 94° C. for 1 minute,42° C. for 2 minutes in and 72° C. for 3 minutes in the given order,then to 35-time cycles of incubating at 94° C. for 1 minute, 60° C. for2 minutes and 72° C. for 3 minutes in the given order to effect PCRreaction.

[0084] Fifty nanograms of the obtained PCR product was added with 1 ngof “pCR-SCRIPT SK(+)”, a plasmid vector commercialized by Takara SyuzoCo. Ltd., Otsu, Shiga, Japan, and reacted using “DNA LIGATION KITVERSION 2”, a DNA ligation kit commercialized by Takara Shuzo Co. Ltd.,Otsu, Shiga, Japan, at 16° C. for 2 hours to insert the DNA fragment asthe PCR product into the plasmid vector. A portion of the reactionproduct was sampled and “XL1-BLUE MRF′ KAN”, a strain of Escherichiacoli commercialized by Stratagene Cloning Systems, California, U.S.A.,was transformed therewith in usual manner.

[0085] The transformant obtained in the above was inoculated in LBmedium (pH 7.5) containing 100 μg/ml ampicillin and cultivated at 37° C.for 18 hours, after which the cells were collected from the culture andtreated in usual manner to obtain the plasmid DNA. After confirming bythe dideoxy method that the plasmid DNA contained the nucleotidesequence of SEQ ID NO: 10, the plasmid DNA was exposed to bothrestriction enzymes NotI and SalI, and 100 ng of the resultant DNAfragment was added with long of “pEF-BOS”, a plasmid vector prepared inaccordance with the method described in S. Mizushima, Nucleic AcidResearch, Vol. 18, No. 17, pp. 5,332 (1990) with slight modification andalso predigested with both restriction enzymes NotI and XhoI, andsubjected to ligation reaction using “LIGATION KIT VERSION 2”, a DNAligation kit commercialized by Takara Shuzo Co., Ltd., Otsu, Shiga,Japan, at 16° C. for 2 hours. A portion of the reaction product wassampled and introduced in usual manner into “XL1-BLUE MRF′ KAN”, astrain of Escherichia coli commercialized by Stratagene Cloning Systems,California, U.S.A., thus obtaining a transformant “EFHIL18R-14” whichcontained a recombinant DNA “pEFHIL18R-14” of this invention. Therecombinant DNA “pEFHIL18R-14” was analyzed in usual manner, revealingthat in the recombinant DNA, a cDNA “EFHIL18R-14 cDNA”, which containedthe nucleotide sequence of SEQ ID NO: 6 encoding the polypeptide of thisinvention, was located downstream the elongation factor 1 promotor EF1αPas shown in FIG. 7.

Example 6-2 Preparation of Transformant

[0086] A transformant “EFHIL18R-14” obtained by the method. in Example6-1 was inoculated in LB medium (pH 7.5) containing 100 μg/ml ampicillinand cultivated at 37° C. for 18 hours, after which the cells werecollected from the culture and treated in usual manner to obtain theplasmid DNA. Separately, COS-1 cell (ATCC CRL-1650), a fibroblastoidcell line derived from a kidney of African green monkey, wasproliferated in usual manner, and 20 micrograms of the plasmid DNAobtained in the above was introduced by conventional electroporationmethod into 1×10⁷ COS-1 cells to obtain transformant cells whichcontained the DNA of this invention.

Example 6-3 Preparation of Soluble Polypeptide

[0087] “ASF104”, a serum-free nutrient culture medium commercialized byAjinomoto Co., Inc., Tokyo, Japan, was distributed in flat-bottomedculture bottles, inoculated with ransformant cells, obtained by themethod in Example 6-2, to givee a cell density of 1×10⁵ cells/ml, andcultured in usual manner at 37° C. in 5 v/v % CO₂ incubator for 3 days.The supernatant was collected from the culture and charged to a columnof an immobilized monoclonal antibody #117-10C prepared by the method inExample 1-5, after which the column was applied first with PBScontaining 12 mM CHAPS to remove non-adsorbed components, then with 35mM ethylamine (pH 10.8) containing 2 mM CHAPS while collecting andfractionating the eluate. Each fraction was checked for presence ofhuman soluble polypeptide by the method in Example 1-1 using¹²⁵I-labelled human IL-18, selected and pooled to obtain per 10⁸starting cells about 2 ml of an aqueous solution which contained apolypeptide with the amino acid sequence of SEQ ID NO: 22. The proteincontent in the solution was about 10 μg/ml.

[0088] The soluble polypeptide thus obtained was studied forphysicochemical properties by the method in Example 1. As the result,the soluble polypeptide obtained in this Example contained each aminoacid sequences in SEQ ID NOs: 12 to 17 and 19 as partial sequences, aswell as exhibiting physiological activities which were similar to theIL-18R from L428 cell.

EXAMPLE 7 Soluble Polypeptide of Human Origin

[0089] One nanogram of an recombinant DNA “pEFHIL18R-14” obtained by themethod in Example 6-1, 10 μl of 10×PCR buffer and 1 μl of 25 mM dNTP mixwere placed in 0.5 ml-reaction tube, added with 1 μl of 2.5units/μl PfuDNA polymerase, further added with appropriate amounts ofoligonucleotides as sense and antisense primers having respectivenucleotide sequences as shown with 5′-TCAGTCGACGCCACCATGAATTGTAGAG-3′and 5′-GAAGCGGCCGCTCATTAGTGATGGTGATGGTGATGTGCAACATGGTTAAGCTT-3′, andfilled up to 100 μl with sterile distilled water. The resultant mixturewas subjected first to 3-time cycles of incubating at 94° C. for 1minute, 42° C. for 2 minutes and 72° C. for 1 minute in the given order,then to 35-time cycles of incubating at 94° C. for 1 minute, 64° C. for1 minute and 72° C. for 1 minute in the given order to effect PCRreaction, thus obtaining a DNA fragment which consisted of thenucleotide sequence of SEQ ID NO: 5, a digestion site for restrictionenzyme SalI and a Kozak's sequence both linked to the 5′-terminal of thenucleotide sequence of SEQ ID NO: 5, and a digestion site forrestriction enzyme NotI and a nucleotide sequence encoding (His)₆ tagboth linked to the 3′-terminal of the nucleotide sequence of SEQ ID NO:5. This DNA fragment was introduced similarly as in Example 6-1 in“XL1-Blue MRF′ Kan”, a strain of Escherichia coli commercialized byStratagene Cloning Systems, California, U.S.A., to obtain a transformantwhich contained a recombinant DNA “pEFHIL18RD1-2-H” according to thisinvention. Analysis of the recombinant DNA in usual manner confirmedthat in this recombinant DNA a cDNA “HIL18RD1-2-H”, which contained thenucleotide sequence of SEQ ID NO: 5 encoding the polypeptide of thisinvention, was located downstream the elongation factor promotor EF1αPas shown in FIG. 8.

[0090] The recombinant DNA “pEFHIL18RD1-2-H” was introduced in COS-1cells similarly as in Example 6-2 using the transformant thus obtained,and the COS-1 cells were then cultivated similarly as in Example 6-3.The supernatant of the resultant culture was concentrated with membranefiltration, and charged on a column of “Ni-NTA Spin Kit”, a gel productfor affinity chromatography commercialized by QIAGEN GmbH, Hilden,Germany, which was then applied with PBS containing 20 mM imidazole toremove the non-adsorbed fractions. Thereafter, the column was appliedwith PBS containing 250 mM imidazole, and the eluate was collected infractions while checking the presence of human soluble polypeptide ineach fraction by the method in Example 1-1 using ¹²⁵I-labelled humanIL-18, after which the fractions with the polypeptide were collected andpooled, thus obtaining about 2 ml of an aqueous solution containing thepolypeptide with the amino acid sequence of SEQ ID NO: 23 per starting10⁸ cells. The protein content in the solution was about 10 μg/ml.

[0091] The soluble polypeptide thus obtained was studied forphysicochemical properties by the method in Example 1. As the result,the soluble polypeptide obtained in this Example contained a part orwhole of each amino acid sequences in SEQ ID NOs: 14 to 16 and 19 aspartial amino acid sequences, as well as exhibiting physiologicalactivities which were similar to those of IL-18R from L428 cell.

EXAMPLE 8 Soluble Polypeptide of Human Origin

[0092] A transformant containing a recombinant DNA “pEFHIL18RD1-H”according to this invention was prepared similarly as in Example 7,except that sense and antisense primers were replaced witholigonucleotides having respective nucleotide sequences as shown with5′-TCAGTCGACGCCACCATGAATTGTAGAG-3′ and 5′-GAAGCGGCCGCTCATTAGTGATGGTGATGGTGATGTCTTTCAGTGAAACAGCT-3′. Analysis ofthe recombinant DNA in usual manner confirmed that in the recombinantDNA a cDNA “HIL18RD1-H”, which contained the nucleotide sequence of SEQID NO: 3 encoding the polypeptide of this invention, was locateddownstream the elongation factor promotor EF1αP as shown in FIG. 9.Thereafter, similarly as in Example 7, the recombinant DNA wasintroduced in COS-1 cells and brought into expression, thus obtainingabout 2 ml of an aqueous solution containing a polypeptide with theamino acid sequence of SEQ ID NO: 24 per 10⁸ starting cells. The proteincontent in the solution was about 10 μg/ml.

[0093] The polypeptide of this invention thus obtained were studied forphysicochemical properties by the method in Example 1. As the result,the soluble polypeptide obtained in this Example contained each aminoacid sequences of SEQ ID NOs: 14 and 15 as partial amino acid sequences,as well as exhibiting physiological activities which were similar tothose of the IL-18R from L428 cell.

EXAMPLE 9 Soluble Polypeptide of Mouse Origin Example 9-1 Preparation ofRecombinant DNA

[0094] A reaction product containing a first strand cDNA was obtained bysubjecting an mRNA, prepared in usual manner from mouse liver, in placewith that from L428 cell to the same reaction to synthesize first strandcDNA as in Example 2-3. The reaction product was treated by the same PCRmethod. as in Example 2-3, except that the sense and antisense primerswere replaced with oligonucleotides having respective nucleotidesequence as shown with 5′-TCAGTCGACGCCACCATGCATCATGAAGAA-3′ and5′-GAAGCGGCCGCATCATTAGTGATGGTGATGGTGATGTGTAAAGACATGGCC-3′, which hadbeen prepared on the basis of the amino acid sequence described in P.Parnet et al., The Journal of Biological Chemistry, Vol. 271, pp.3,967-3,970 (1996) and also the nucleotide sequence of SEQ ID NO: 1:This operation gave a DNA fragment which comprised the nucleotidesequence of SEQ ID NO: 11, a digestion site for restriction enzyme SalIlinked to the 5′-terminal in the nucleotide sequence of the SEQ ID NO:11, and a cleavage site for restriction enzyme NotI and a nucleotidesequence encoding (His)₆ tag both linked to the 3′-terminal in thenucleotide sequence of the SEQ ID NO: 11.

[0095] According to the method in Example 6-1, this DNA fragment wasintroduced into “XL1-BLUE MRF′ KAN”, a strain of Escherichia colicommercialized by Stratagene Cloning Systems, California, U.S.A., totransform. After a plasmid DNA was collected from the transformant andconfirmed to contain the nucleotide sequence of SEQ ID NO: 11, theplasmid DNA was introduced into “XL1-BLUE MRF′ KAN”, a strain ofEscherichia coli strain commercialized by Stratagene Cloning Systems,California, U.S.A., to obtain a transformant “EFMIL18RSHT” whichcontains a recombinant DNA “pEFMIL18RSHT” according to this invention.Analysis in usual manner confirmed that in the recombinant DNA“pEFMIL18RSHT” a cDNA “EFMIL18RSHT cDNA”, which contained the nucleotidesequence of SEQ ID NO: 4 encoding the polypeptide of this invention, waslinked to downstream of the elongation factor 1 promotor EF1αP, as shownin FIG. 8.

Example 9-2 Preparation of Transformant and Soluble Polypeptide

[0096] According to the method in Example 6-2, a plasmid DNA wascollected from a transformant “EFMIL18RSHT” obtained by the method inExample 9-1, and introduced into COS-1 cells to obtain transformantcells which contained a DNA encoding a soluble polypeptide of mouseorigin.

[0097] “ASF104”, a serum-free nutrient culture medium commercialized byAjinomoto Co., Inc., Tokyo, Japan, was distributed in flat-bottomedculture bottles, inoculated with the transformed COS-1 cells to give acell density of 1×10⁵cells/ml, and cultivated in usual manner at 37° C.in 5 v/v % CO₂ incubator for 3 days. The supernatant was collected fromthe resultant culture and charged to a column of “Ni-NTA”, a gel productfor affinity chromatography, commercialized by QIAGEN GmbH, Hilden,Germany, after which the column was applied first with PBS containing 20mM imidazole to remove non-adsorbed components, then with PBS containing250 mM imidazole while collecting and fractionating the eluate. Thefractions were checked for presence of mouse soluble polypeptide by themethod in Example 1-1 using ¹²⁵I-labelled mouse IL-18, selected andpooled, thus obtaining per 10⁸ starting cells about 2 ml of an aqueoussolution which contained a polypeptide with the amino acid sequence ofSEQ ID NO: 25. The protein content in the solution was about 100 μg/ml.The soluble polypeptide thus obtained was studied in accordance with themethod in Example 1, revealing that it efficiently neutralized mouseIL-18.

EXAMPLE 10 Liquid Agent

[0098] Either polypeptide obtained by the method in Examples 5 to 8 wasseparately dissolved in aliquots of physiological saline containing asstabilizer 1 w/v % “TREHAOSE”, a powdered crystalline trehalosecommercialized by Hayashibara Co., Ltd., Okayama, Japan, to giverespective concentration of 1 mg/ml, and the resultant mixtures wereseparately and sterilely filtered with membrane in usual manner toobtain four distinct liquid agents.

[0099] The products, which are excellent in stability, are useful asinjection, ophthalmic solution and collunarium in treatment andprevention of susceptive diseases including autoimmune diseases.

EXAMPLE 11 Dried Injection

[0100] One hundred milligrams of either polypeptide obtained by themethods in Example 5 to 8 was separately dissolved in aliquots ofphysiological saline containing 1 w/v % sucrose as stabilizer, theresultant solutions were separately and sterilely filtered withmembrane, distributed in vials in every 1 ml aliquot, lyophilized andsealed in usual manner to obtain four distinct pulverized agents.

[0101] The products, which are excellent in stability, are useful asdried injection in treatment and prevention of susceptive diseasesincluding autoimmune diseases.

EXAMPLE 12 Ointment

[0102] “HI-BIS-WAKO 104”, a carboxyvinylpolymer commercialized by WakoPure Chemicals, Tokyo, Japan, and “TREHAOSE”, a powdered crystallinetrehalose commercialized by Hayashibara Co., Ltd., Okayama, Japan, weredissolved in sterilized distilled water to give respectiveconcentrations of 1.4 w/w % and 2.0 w/w %, and either polypeptideobtained by the methods in Examples 5 to 8 was separately mixed withaliquots of the resultant solution to homogeneity, and adjusted to pH7.2 to obtain four distinct paste agents containing about 1 mg/g of thepolypeptide of this invention each.

[0103] The products, which are excellent in both spreadablity andstability, are useful as ointment in treatment and prevention ofsusceptive diseases including autoimmune diseases.

EXAMPLE 13 Tablet

[0104] Aliquots of “FINETOSE”, a pulverized anhydrous crystallinealpha-maltose commercialized by Hayashibara Co., Ltd., Okayama, Japan,were separately admixed with either polypeptide, obtained by the methodsin Examples 5 to 8, and aliquots of “LUMIN” as cell activator,[bis-4-(1-ethylquinoline)][γ-4′-(1-ethylquinoline)] pentamethioninecyanine, to homogeneity, and the resultant mixtures were separatelytableted in usual manner to obtain four distinct types of tablets, about200 mg each, containing about 1 mg/tablet of the polypeptide of thisinvention and also 1 mg/tablet of LUMIN each.

[0105] The products, which are excellent in swallowability and stabilityand also bears an cell activating property, are useful as tablet intreatment and prevention of susceptive diseases including autoimmunediseases.

EXPERIMENT Acute Toxicity Test

[0106] In usual manner, a variety of agents, obtained by the methods inExamples 8 to 11, were percutaneously or orally administrated orintraperitoneally injected to 8 week-old mice. As the result, the LD₅₀of each sample was proved about 1 mg or higher per body weight of mousein terms of the amount of the polypeptide, regardless of administrationroute. This does support that the polypeptide of this invention is safewhen incorporated in pharmaceuticals directed to use in mammalsincluding human.

[0107] As explained above, this invention is based on the discovery of anovel receptor protein which recognizes IL-18. The polypeptide of thisinvention exhibits a remarkable efficacy in relief of rejection reactionassociated with grafts of organs and also in treatment and prevention ofvarious disease resulting from excessive immunoreaction because thepolypeptide bears properties of suppressing and regulatingimmunoreaction in mammals including human. Further, the polypeptide ofthis invention is useful in clarification of physiological activities ofIL-18, establishment of hybridoma cells which are capable of producingmonoclonal antibodies specific to IL-18R, and also affinitychromatography and labelled assay to purify and detect IL-18. Inaddition, the polypeptide of this invention, in particular, that insoluble form is useful in screening in vivo and in vitro agonists andantagonists to IL-18. The polypeptide of this invention, which bearsthese outstanding usefulness, can be easily prepared in desired amountsby the process according to this invention using recombinant DNAtechniques.

[0108] This invention, which exhibits these remarkable effects, would bevery significant and contributive to the art.

0 SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES:37 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 1563 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A)NAME/KEY:mat peptide (B) LOCATION:1..1563 (C) IDENTIFICATION METHOD:E(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GAA TCT TGT ACT TCA CGT CCC CACATT ACT GTG GTT GAA GGG GAA CCT 48 Glu Ser Cys Thr Ser Arg Pro His IleThr Val Val Glu Gly Glu Pro 1 5 10 15 TTC TAT CTG AAA CAT TGC TCG TGTTCA CTT GCA CAT GAG ATT GAA ACA 96 Phe Tyr Leu Lys His Cys Ser Cys SerLeu Ala His Glu Ile Glu Thr 20 25 30 ACC ACC AAA AGC TGG TAC AAA AGC AGTGGA TCA CAG GAA CAT GTG GAG 144 Thr Thr Lys Ser Trp Tyr Lys Ser Ser GlySer Gln Glu His Val Glu 35 40 45 CTG AAC CCA AGG AGT TCC TCG AGA ATT GCTTTG CAT GAT TGT GTT TTG 192 Leu Asn Pro Arg Ser Ser Ser Arg Ile Ala LeuHis Asp Cys Val Leu 50 55 60 GAG TTT TGG CCA GTT GAG TTG AAT GAC ACA GGATCT TAC TTT TTC CAA 240 Glu Phe Trp Pro Val Glu Leu Asn Asp Thr Gly SerTyr Phe Phe Gln 65 70 75 80 ATG AAA AAT TAT ACT CAG AAA TGG AAA TTA AATGTC ATC AGA AGA AAT 288 Met Lys Asn Tyr Thr Gln Lys Trp Lys Leu Asn ValIle Arg Arg Asn 85 90 95 AAA CAC AGC TGT TTC ACT GAA AGA CAA GTA ACT AGTAAA ATT GTG GAA 336 Lys His Ser Cys Phe Thr Glu Arg Gln Val Thr Ser LysIle Val Glu 100 105 110 GTT AAA AAA TTT TTT CAG ATA ACC TGT GAA AAC AGTTAC TAT CAA ACA 384 Val Lys Lys Phe Phe Gln Ile Thr Cys Glu Asn Ser TyrTyr Gln Thr 115 120 125 CTG GTC AAC AGC ACA TCA TTG TAT AAG AAC TGT AAAAAG CTA CTA CTG 432 Leu Val Asn Ser Thr Ser Leu Tyr Lys Asn Cys Lys LysLeu Leu Leu 130 135 140 GAG AAC AAT AAA AAC CCA ACG ATA AAG AAG AAC GCCGAG TTT GAA GAT 480 Glu Asn Asn Lys Asn Pro Thr Ile Lys Lys Asn Ala GluPhe Glu Asp 145 150 155 160 CAG GGG TAT TAC TCC TGC GTG CAT TTC CTT CATCAT AAT GGA AAA CTA 528 Gln Gly Tyr Tyr Ser Cys Val His Phe Leu His HisAsn Gly Lys Leu 165 170 175 TTT AAT ATC ACC AAA ACC TTC AAT ATA ACA ATAGTG GAA GAT CGC AGT 576 Phe Asn Ile Thr Lys Thr Phe Asn Ile Thr Ile ValGlu Asp Arg Ser 180 185 190 AAT ATA GTT CCG GTT CTT CTT GGA CCA AAG CTTAAC CAT GTT GCA GTG 624 Asn Ile Val Pro Val Leu Leu Gly Pro Lys Leu AsnHis Val Ala Val 195 200 205 GAA TTA GGA AAA AAC GTA AGG CTC AAC TGC TCTGCT TTG CTG AAT GAA 672 Glu Leu Gly Lys Asn Val Arg Leu Asn Cys Ser AlaLeu Leu Asn Glu 210 215 220 GAG GAT GTA ATT TAT TGG ATG TTC GGG GAA GAAAAT GGA TCG GAT CCT 720 Glu Asp Val Ile Tyr Trp Met Phe Gly Glu Glu AsnGly Ser Asp Pro 225 230 235 240 AAT ATA CAT GAA GAG AAA GAA ATG AGA ATTATG ACT CCA GAA GGC AAA 768 Asn Ile His Glu Glu Lys Glu Met Arg Ile MetThr Pro Glu Gly Lys 245 250 255 TGG CAT GCT TCA AAA GTA TTG AGA ATT GAAAAT ATT GGT GAA AGC AAT 816 Trp His Ala Ser Lys Val Leu Arg Ile Glu AsnIle Gly Glu Ser Asn 260 265 270 CTA AAT GTT TTA TAT AAT TGC ACT GTG GCCAGC ACG GGA GGC ACA GAC 864 Leu Asn Val Leu Tyr Asn Cys Thr Val Ala SerThr Gly Gly Thr Asp 275 280 285 ACC AAA AGC TTC ATC TTG GTG AGA AAA GACATG GCT GAT ATC CCA GGC 912 Thr Lys Ser Phe Ile Leu Val Arg Lys Asp MetAla Asp Ile Pro Gly 290 295 300 CAC GTC TTC ACA AGA GGA ATG ATC ATA GCTGTT TTG ATC TTG GTG GCA 960 His Val Phe Thr Arg Gly Met Ile Ile Ala ValLeu Ile Leu Val Ala 305 310 315 320 GTA GTG TGC CTA GTG ACT GTG TGT GTCATT TAT AGA GTT GAC TTG GTT 1008 Val Val Cys Leu Val Thr Val Cys Val IleTyr Arg Val Asp Leu Val 325 330 335 CTA TTT TAT AGA CAT TTA ACG AGA AGAGAT GAA ACA TTA ACA GAT GGA 1056 Leu Phe Tyr Arg His Leu Thr Arg Arg AspGlu Thr Leu Thr Asp Gly 340 345 350 AAA ACA TAT GAT GCT TTT GTG TCT TACCTA AAA GAA TGC CGA CCT GAA 1104 Lys Thr Tyr Asp Ala Phe Val Ser Tyr LeuLys Glu Cys Arg Pro Glu 355 360 365 AAT GGA GAG GAG CAC ACC TTT GCT GTGGAG ATT TTG CCC AGG GTG TTG 1152 Asn Gly Glu Glu His Thr Phe Ala Val GluIle Leu Pro Arg Val Leu 370 375 380 GAG AAA CAT TTT GGG TAT AAG TTA TGCATA TTT GAA AGG GAT GTA GTG 1200 Glu Lys His Phe Gly Tyr Lys Leu Cys IlePhe Glu Arg Asp Val Val 385 390 395 400 CCT GGA GGA GCT GTT GTT GAT GAAATC CAC TCA CTG ATA GAG AAA AGC 1248 Pro Gly Gly Ala Val Val Asp Glu IleHis Ser Leu Ile Glu Lys Ser 405 410 415 CGA AGA CTA ATC ATT GTC CTA AGTAAA AGT TAT ATG TCT AAT GAG GTC 1296 Arg Arg Leu Ile Ile Val Leu Ser LysSer Tyr Met Ser Asn Glu Val 420 425 430 AGG TAT GAA CTT GAA AGT GGA CTCCAT GAA GCA TTG GTG GAA AGA AAA 1344 Arg Tyr Glu Leu Glu Ser Gly Leu HisGlu Ala Leu Val Glu Arg Lys 435 440 445 ATT AAA ATA ATC TTA ATT GAA TTTACA CCT GTT ACT GAC TTC ACA TTC 1392 Ile Lys Ile Ile Leu Ile Glu Phe ThrPro Val Thr Asp Phe Thr Phe 450 455 460 TTG CCC CAA TCA CTA AAG CTT TTGAAA TCT CAC AGA GTT CTG AAG TGG 1440 Leu Pro Gln Ser Leu Lys Leu Leu LysSer His Arg Val Leu Lys Trp 465 470 475 480 AAG GCC GAT AAA TCT CTT TCTTAT AAC TCA AGG TTC TGG AAG AAC CTT 1488 Lys Ala Asp Lys Ser Leu Ser TyrAsn Ser Arg Phe Trp Lys Asn Leu 485 490 495 CTT TAC TTA ATG CCT GCA AAAACA GTC AAG CCA GGT AGA GAC GAA CCG 1536 Leu Tyr Leu Met Pro Ala Lys ThrVal Lys Pro Gly Arg Asp Glu Pro 500 505 510 GAA GTC TTG CCT GTT CTT TCCGAG TCT 1563 Glu Val Leu Pro Val Leu Ser Glu Ser 515 520 (2) INFORMATIONFOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1557 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY:linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY:mat peptide(B) LOCATION:1..1557 (C) IDENTIFICATION METHOD:S (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 2: TCA AAA AGT TGT ATT CAC CGA TCA CAA ATT CATGTG GTA GAG GGA GAA 48 Ser Lys Ser Cys Ile His Arg Ser Gln Ile His ValVal Glu Gly Glu 1 5 10 15 CCT TTT TAT CTG AAG CCA TGT GGC ATA TCT GCACCA GTG CAC AGG AAT 96 Pro Phe Tyr Leu Lys Pro Cys Gly Ile Ser Ala ProVal His Arg Asn 20 25 30 GAA ACA GCC ACC ATG AGA TGG TTC AAA GGC AGT GCTTCA CAT GAG TAT 144 Glu Thr Ala Thr Met Arg Trp Phe Lys Gly Ser Ala SerHis Glu Tyr 35 40 45 AGA GAG CTG AAC AAC AGA AGC TCG CCC AGA GTC ACT TTTCAT GAT CAC 192 Arg Glu Leu Asn Asn Arg Ser Ser Pro Arg Val Thr Phe HisAsp His 50 55 60 ACC TTG GAA TTC TGG CCA GTT GAG ATG GAG GAT GAG GGA ACGTAC ATT 240 Thr Leu Glu Phe Trp Pro Val Glu Met Glu Asp Glu Gly Thr TyrIle 65 70 75 80 TCT CAA GTC GGA AAT GAT CGT CGC AAT TGG ACC TTA AAT GTCACC AAA 288 Ser Gln Val Gly Asn Asp Arg Arg Asn Trp Thr Leu Asn Val ThrLys 85 90 95 AGA AAC AAA CAC AGC TGT TTC TCT GAC AAG CTC GTG ACA AGC AGAGAT 336 Arg Asn Lys His Ser Cys Phe Ser Asp Lys Leu Val Thr Ser Arg Asp100 105 110 GTT GAA GTT AAC AAA TCT CTG CAT ATC ACT TGT AAG AAT CCT AACTAT 384 Val Glu Val Asn Lys Ser Leu His Ile Thr Cys Lys Asn Pro Asn Tyr115 120 125 GAA GAG CTG ATC CAG GAC ACA TGG CTG TAT AAG AAC TGT AAG GAAATA 432 Glu Glu Leu Ile Gln Asp Thr Trp Leu Tyr Lys Asn Cys Lys Glu Ile130 135 140 TCC AAA ACC CCA AGG ATC CTG AAG GAT GCC GAG TTT GGA GAT GAGGGC 480 Ser Lys Thr Pro Arg Ile Leu Lys Asp Ala Glu Phe Gly Asp Glu Gly145 150 155 160 TAC TAC TCC TGC GTG TTT TCT GTC CAC CAT AAT GGG ACA CGGTAC AAC 528 Tyr Tyr Ser Cys Val Phe Ser Val His His Asn Gly Thr Arg TyrAsn 165 170 175 ATC ACC AAG ACT GTC AAT ATA ACA GTT ATT GAA GGA AGG AGTAAA GTA 576 Ile Thr Lys Thr Val Asn Ile Thr Val Ile Glu Gly Arg Ser LysVal 180 185 190 ACT CCA GCT ATT TTA GGA CCA AAG TGT GAG AAG GTT GGT GTAGAA CTA 624 Thr Pro Ala Ile Leu Gly Pro Lys Cys Glu Lys Val Gly Val GluLeu 195 200 205 GGA AAG GAT GTG GAG TTG AAC TGC AGT GCT TCA TTG AAT AAAGAC GAT 672 Gly Lys Asp Val Glu Leu Asn Cys Ser Ala Ser Leu Asn Lys AspAsp 210 215 220 CTG TTT TAT TGG AGC ATC AGG AAA GAG GAC AGC TCA GAC CCTAAT GTG 720 Leu Phe Tyr Trp Ser Ile Arg Lys Glu Asp Ser Ser Asp Pro AsnVal 225 230 235 240 CAA GAA GAC AGG AAG GAG ACG ACA ACA TGG ATT TCT GAAGGC AAA CTG 768 Gln Glu Asp Arg Lys Glu Thr Thr Thr Trp Ile Ser Glu GlyLys Leu 245 250 255 CAT GCT TCA AAA ATA CTG AGA TTT CAG AAA ATT ACT GAAAAC TAT CTC 816 His Ala Ser Lys Ile Leu Arg Phe Gln Lys Ile Thr Glu AsnTyr Leu 260 265 270 AAT GTT TTA TAT AAT TGC ACC GTG GCC AAC GAA GAA GCCATA GAC ACC 864 Asn Val Leu Tyr Asn Cys Thr Val Ala Asn Glu Glu Ala IleAsp Thr 275 280 285 AAG AGC TTC GTC TTG GTG AGA AAA GAA ATA CCT GAT ATCCCA GGC CAT 912 Lys Ser Phe Val Leu Val Arg Lys Glu Ile Pro Asp Ile ProGly His 290 295 300 GTC TTT ACA GGA GGA GTA ACT GTG CTT GTT CTC GCC TCTGTG GCA GCA 960 Val Phe Thr Gly Gly Val Thr Val Leu Val Leu Ala Ser ValAla Ala 305 310 315 320 GTG TGT ATA GTG ATT TTG TGT GTC ATT TAT AAA GTTGAC TTG GTT CTG 1008 Val Cys Ile Val Ile Leu Cys Val Ile Tyr Lys Val AspLeu Val Leu 325 330 335 TTC TAT AGG CGC ATA GCG GAA AGA GAC GAG ACA CTAACA GAT GGT AAA 1056 Phe Tyr Arg Arg Ile Ala Glu Arg Asp Glu Thr Leu ThrAsp Gly Lys 340 345 350 ACA TAT GAT GCC TTT GTG TCT TAC CTG AAA GAG TGTCAT CCT GAG AAT 1104 Thr Tyr Asp Ala Phe Val Ser Tyr Leu Lys Glu Cys HisPro Glu Asn 355 360 365 AAA GAA GAG TAT ACT TTT GCT GTG GAG ACG TTA CCCAGG GTC CTG GAG 1152 Lys Glu Glu Tyr Thr Phe Ala Val Glu Thr Leu Pro ArgVal Leu Glu 370 375 380 AAA CAG TTT GGG TAT AAG TTA TGC ATA TTT GAA AGAGAT GTG GTG CCT 1200 Lys Gln Phe Gly Tyr Lys Leu Cys Ile Phe Glu Arg AspVal Val Pro 385 390 395 400 GGC GGA GCT GTT GTC GAG GAG ATC CAT TCA CTGATA GAG AAA AGC CGG 1248 Gly Gly Ala Val Val Glu Glu Ile His Ser Leu IleGlu Lys Ser Arg 405 410 415 AGG CTA ATC ATC GTT CTC AGC CAG AGT TAC CTGACT AAC GGA GCC AGG 1296 Arg Leu Ile Ile Val Leu Ser Gln Ser Tyr Leu ThrAsn Gly Ala Arg 420 425 430 CGT GAG CTC GAG AGT GGA CTC CAC GAA GCA CTGGTA GAG AGG AAG ATT 1344 Arg Glu Leu Glu Ser Gly Leu His Glu Ala Leu ValGlu Arg Lys Ile 435 440 445 AAG ATC ATC TTA ATT GAG TTT ACT CCA GCC AGCAAC ATC ACC TTT CTC 1392 Lys Ile Ile Leu Ile Glu Phe Thr Pro Ala Ser AsnIle Thr Phe Leu 450 455 460 CCC CCG TCG CTG AAA CTC CTG AAG TCC TAC AGAGTT CTA AAA TGG AGG 1440 Pro Pro Ser Leu Lys Leu Leu Lys Ser Tyr Arg ValLeu Lys Trp Arg 465 470 475 480 GCT GAC AGT CCC TCC ATG AAC TCA AGG TTCTGG AAG AAT CTT GTT TAC 1488 Ala Asp Ser Pro Ser Met Asn Ser Arg Phe TrpLys Asn Leu Val Tyr 485 490 495 CTG ATG CCC GCA AAA GCC GTC AAG CCA TGGAGA GAG GAG TCG GAG GCG 1536 Leu Met Pro Ala Lys Ala Val Lys Pro Trp ArgGlu Glu Ser Glu Ala 500 505 510 CGG TCT GTT CTC TCA GCA CCT 1557 Arg SerVal Leu Ser Ala Pro 515 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 312 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix)FEATURE: (A) NAME/KEY:mat peptide (B) LOCATION:1..312 (C) IDENTIFICATIONMETHOD:S (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GAA TCT TGT ACT TCACGT CCC CAC ATT ACT GTG GTT GAA GGG GAA CCT 48 Glu Ser Cys Thr Ser ArgPro His Ile Thr Val Val Glu Gly Glu Pro 1 5 10 15 TTC TAT CTG AAA CATTGC TCG TGT TCA CTT GCA CAT GAG ATT GAA ACA 96 Phe Tyr Leu Lys His CysSer Cys Ser Leu Ala His Glu Ile Glu Thr 20 25 30 ACC ACC AAA AGC TGG TACAAA AGC AGT GGA TCA CAG GAA CAT GTG GAG 144 Thr Thr Lys Ser Trp Tyr LysSer Ser Gly Ser Gln Glu His Val Glu 35 40 45 CTG AAC CCA AGG AGT TCC TCGAGA ATT GCT TTG CAT GAT TGT GTT TTG 192 Leu Asn Pro Arg Ser Ser Ser ArgIle Ala Leu His Asp Cys Val Leu 50 55 60 GAG TTT TGG CCA GTT GAG TTG AATGAC ACA GGA TCT TAC TTT TTC CAA 240 Glu Phe Trp Pro Val Glu Leu Asn AspThr Gly Ser Tyr Phe Phe Gln 65 70 75 80 ATG AAA AAT TAT ACT CAG AAA TGGAAA TTA AAT GTC ATC AGA AGA AAT 288 Met Lys Asn Tyr Thr Gln Lys Trp LysLeu Asn Val Ile Arg Arg Asn 85 90 95 AAA CAC AGC TGT TTC ACT GAA AGA 312Lys His Ser Cys Phe Thr Glu Arg 100 (2) INFORMATION FOR SEQ ID NO: 4:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 921 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULETYPE: cDNA (ix) FEATURE: (A) NAME/KEY:mat peptide (B) LOCATION:1..921(C) IDENTIFICATION METHOD:S (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: TCAAAA AGT TGT ATT CAC CGA TCA CAA ATT CAT GTG GTA GAG GGA GAA 48 Ser LysSer Cys Ile His Arg Ser Gln Ile His Val Val Glu Gly Glu 1 5 10 15 CCTTTT TAT CTG AAG CCA TGT GGC ATA TCT GCA CCA GTG CAC AGG AAT 96 Pro PheTyr Leu Lys Pro Cys Gly Ile Ser Ala Pro Val His Arg Asn 20 25 30 GAA ACAGCC ACC ATG AGA TGG TTC AAA GGC AGT GCT TCA CAT GAG TAT 144 Glu Thr AlaThr Met Arg Trp Phe Lys Gly Ser Ala Ser His Glu Tyr 35 40 45 AGA GAG CTGAAC AAC AGA AGC TCG CCC AGA GTC ACT TTT CAT GAT CAC 192 Arg Glu Leu AsnAsn Arg Ser Ser Pro Arg Val Thr Phe His Asp His 50 55 60 ACC TTG GAA TTCTGG CCA GTT GAG ATG GAG GAT GAG GGA ACG TAC ATT 240 Thr Leu Glu Phe TrpPro Val Glu Met Glu Asp Glu Gly Thr Tyr Ile 65 70 75 80 TCT CAA GTC GGAAAT GAT CGT CGC AAT TGG ACC TTA AAT GTC ACC AAA 288 Ser Gln Val Gly AsnAsp Arg Arg Asn Trp Thr Leu Asn Val Thr Lys 85 90 95 AGA AAC AAA CAC AGCTGT TTC TCT GAC AAG CTC GTG ACA AGC AGA GAT 336 Arg Asn Lys His Ser CysPhe Ser Asp Lys Leu Val Thr Ser Arg Asp 100 105 110 GTT GAA GTT AAC AAATCT CTG CAT ATC ACT TGT AAG AAT CCT AAC TAT 384 Val Glu Val Asn Lys SerLeu His Ile Thr Cys Lys Asn Pro Asn Tyr 115 120 125 GAA GAG CTG ATC CAGGAC ACA TGG CTG TAT AAG AAC TGT AAG GAA ATA 432 Glu Glu Leu Ile Gln AspThr Trp Leu Tyr Lys Asn Cys Lys Glu Ile 130 135 140 TCC AAA ACC CCA AGGATC CTG AAG GAT GCC GAG TTT GGA GAT GAG GGC 480 Ser Lys Thr Pro Arg IleLeu Lys Asp Ala Glu Phe Gly Asp Glu Gly 145 150 155 160 TAC TAC TCC TGCGTG TTT TCT GTC CAC CAT AAT GGG ACA CGG TAC AAC 528 Tyr Tyr Ser Cys ValPhe Ser Val His His Asn Gly Thr Arg Tyr Asn 165 170 175 ATC ACC AAG ACTGTC AAT ATA ACA GTT ATT GAA GGA AGG AGT AAA GTA 576 Ile Thr Lys Thr ValAsn Ile Thr Val Ile Glu Gly Arg Ser Lys Val 180 185 190 ACT CCA GCT ATTTTA GGA CCA AAG TGT GAG AAG GTT GGT GTA GAA CTA 624 Thr Pro Ala Ile LeuGly Pro Lys Cys Glu Lys Val Gly Val Glu Leu 195 200 205 GGA AAG GAT GTGGAG TTG AAC TGC AGT GCT TCA TTG AAT AAA GAC GAT 672 Gly Lys Asp Val GluLeu Asn Cys Ser Ala Ser Leu Asn Lys Asp Asp 210 215 220 CTG TTT TAT TGGAGC ATC AGG AAA GAG GAC AGC TCA GAC CCT AAT GTG 720 Leu Phe Tyr Trp SerIle Arg Lys Glu Asp Ser Ser Asp Pro Asn Val 225 230 235 240 CAA GAA GACAGG AAG GAG ACG ACA ACA TGG ATT TCT GAA GGC AAA CTG 768 Gln Glu Asp ArgLys Glu Thr Thr Thr Trp Ile Ser Glu Gly Lys Leu 245 250 255 CAT GCT TCAAAA ATA CTG AGA TTT CAG AAA ATT ACT GAA AAC TAT CTC 816 His Ala Ser LysIle Leu Arg Phe Gln Lys Ile Thr Glu Asn Tyr Leu 260 265 270 AAT GTT TTATAT AAT TGC ACC GTG GCC AAC GAA GAA GCC ATA GAC ACC 864 Asn Val Leu TyrAsn Cys Thr Val Ala Asn Glu Glu Ala Ile Asp Thr 275 280 285 AAG AGC TTCGTC TTG GTG AGA AAA GAA ATA CCT GAT ATC CCA GGC CAT 912 Lys Ser Phe ValLeu Val Arg Lys Glu Ile Pro Asp Ile Pro Gly His 290 295 300 GTC TTT ACA921 Val Phe Thr 305 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 621 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix)FEATURE: (A) NAME/KEY:mat peptide (B) LOCATION:1..621 (C) IDENTIFICATIONMETHOD:S (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GAA TCT TGT ACT TCACGT CCC CAC ATT ACT GTG GTT GAA GGG GAA CCT 48 Glu Ser Cys Thr Ser ArgPro His Ile Thr Val Val Glu Gly Glu Pro 1 5 10 15 TTC TAT CTG AAA CATTGC TCG TGT TCA CTT GCA CAT GAG ATT GAA ACA 96 Phe Tyr Leu Lys His CysSer Cys Ser Leu Ala His Glu Ile Glu Thr 20 25 30 ACC ACC AAA AGC TGG TACAAA AGC AGT GGA TCA CAG GAA CAT GTG GAG 144 Thr Thr Lys Ser Trp Tyr LysSer Ser Gly Ser Gln Glu His Val Glu 35 40 45 CTG AAC CCA AGG AGT TCC TCGAGA ATT GCT TTG CAT GAT TGT GTT TTG 192 Leu Asn Pro Arg Ser Ser Ser ArgIle Ala Leu His Asp Cys Val Leu 50 55 60 GAG TTT TGG CCA GTT GAG TTG AATGAC ACA GGA TCT TAC TTT TTC CAA 240 Glu Phe Trp Pro Val Glu Leu Asn AspThr Gly Ser Tyr Phe Phe Gln 65 70 75 80 ATG AAA AAT TAT ACT CAG AAA TGGAAA TTA AAT GTC ATC AGA AGA AAT 288 Met Lys Asn Tyr Thr Gln Lys Trp LysLeu Asn Val Ile Arg Arg Asn 85 90 95 AAA CAC AGC TGT TTC ACT GAA AGA CAAGTA ACT AGT AAA ATT GTG GAA 336 Lys His Ser Cys Phe Thr Glu Arg Gln ValThr Ser Lys Ile Val Glu 100 105 110 GTT AAA AAA TTT TTT CAG ATA ACC TGTGAA AAC AGT TAC TAT CAA ACA 384 Val Lys Lys Phe Phe Gln Ile Thr Cys GluAsn Ser Tyr Tyr Gln Thr 115 120 125 CTG GTC AAC AGC ACA TCA TTG TAT AAGAAC TGT AAA AAG CTA CTA CTG 432 Leu Val Asn Ser Thr Ser Leu Tyr Lys AsnCys Lys Lys Leu Leu Leu 130 135 140 GAG AAC AAT AAA AAC CCA ACG ATA AAGAAG AAC GCC GAG TTT GAA GAT 480 Glu Asn Asn Lys Asn Pro Thr Ile Lys LysAsn Ala Glu Phe Glu Asp 145 150 155 160 GAG GGG TAT TAC TCC TGC GTG CATTTC CTT CAT CAT AAT GGA AAA CTA 528 Gln Gly Tyr Tyr Ser Cys Val His PheLeu His His Asn Gly Lys Leu 165 170 175 TTT AAT ATC ACC AAA ACC TTC AATATA ACA ATA GTG GAA GAT CGC AGT 576 Phe Asn Ile Thr Lys Thr Phe Asn IleThr Ile Val Glu Asp Arg Ser 180 185 190 AAT ATA GTT CCG GTT CTT CTT GGACCA AAG CTT AAC CAT GTT GCA 621 Asn Ile Val Pro Val Leu Leu Gly Pro LysLeu Asn His Val Ala 195 200 205 (2) INFORMATION FOR SEQ ID NO: 6: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 927 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (ix) FEATURE: (A) NAME/KEY:mat peptide (B) LOCATION:1..927 (C)IDENTIFICATION METHOD:E (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: GAA TCTTGT ACT TCA CGT CCC CAC ATT ACT GTG GTT GAA GGG GAA CCT 48 Glu Ser CysThr Ser Arg Pro His Ile Thr Val Val Glu Gly Glu Pro 1 5 10 15 TTC TATCTG AAA CAT TGC TCG TGT TCA CTT GCA CAT GAG ATT GAA ACA 96 Phe Tyr LeuLys His Cys Ser Cys Ser Leu Ala His Glu Ile Glu Thr 20 25 30 ACC ACC AAAAGC TGG TAC AAA AGC AGT GGA TCA CAG GAA CAT GTG GAG 144 Thr Thr Lys SerTrp Tyr Lys Ser Ser Gly Ser Gln Glu His Val Glu 35 40 45 CTG AAC CCA AGGAGT TCC TCG AGA ATT GCT TTG CAT GAT TGT GTT TTG 192 Leu Asn Pro Arg SerSer Ser Arg Ile Ala Leu His Asp Cys Val Leu 50 55 60 GAG TTT TGG CCA GTTGAG TTG AAT GAC ACA GGA TCT TAC TTT TTC CAA 240 Glu Phe Trp Pro Val GluLeu Asn Asp Thr Gly Ser Tyr Phe Phe Gln 65 70 75 80 ATG AAA AAT TAT ACTCAG AAA TGG AAA TTA AAT GTC ATC AGA AGA AAT 288 Met Lys Asn Tyr Thr GlnLys Trp Lys Leu Asn Val Ile Arg Arg Asn 85 90 95 AAA CAC AGC TGT TTC ACTGAA AGA CAA GTA ACT AGT AAA ATT GTG GAA 336 Lys His Ser Cys Phe Thr GluArg Gln Val Thr Ser Lys Ile Val Glu 100 105 110 GTT AAA AAA TTT TTT CAGATA ACC TGT GAA AAC AGT TAC TAT CAA ACA 384 Val Lys Lys Phe Phe Gln IleThr Cys Glu Asn Ser Tyr Tyr Gln Thr 115 120 125 CTG GTC AAC AGC ACA TCATTG TAT AAG AAC TGT AAA AAG CTA CTA CTG 432 Leu Val Asn Ser Thr Ser LeuTyr Lys Asn Cys Lys Lys Leu Leu Leu 130 135 140 GAG AAC AAT AAA AAC CCAACG ATA AAG AAG AAC GCC GAG TTT GAA GAT 480 Glu Asn Asn Lys Asn Pro ThrIle Lys Lys Asn Ala Glu Phe Glu Asp 145 150 155 160 CAG GGG TAT TAC TCCTGC GTG CAT TTC CTT CAT CAT AAT GGA AAA CTA 528 Gln Gly Tyr Tyr Ser CysVal His Phe Leu His His Asn Gly Lys Leu 165 170 175 TTT AAT ATC ACC AAAACC TTC AAT ATA ACA ATA GTG GAA GAT CGC AGT 576 Phe Asn Ile Thr Lys ThrPhe Asn Ile Thr Ile Val Glu Asp Arg Ser 180 185 190 AAT ATA GTT CCG GTTCTT CTT GGA CCA AAG CTT AAC CAT GTT GCA GTG 624 Asn Ile Val Pro Val LeuLeu Gly Pro Lys Leu Asn His Val Ala Val 195 200 205 GAA TTA GGA AAA AACGTA AGG CTC AAC TGC TCT GCT TTG CTG AAT GAA 672 Glu Leu Gly Lys Asn ValArg Leu Asn Cys Ser Ala Leu Leu Asn Glu 210 215 220 GAG GAT GTA ATT TATTGG ATG TTC GGG GAA GAA AAT GGA TCG GAT CCT 720 Glu Asp Val Ile Tyr TrpMet Phe Gly Glu Glu Asn Gly Ser Asp Pro 225 230 235 240 AAT ATA CAT GAAGAG AAA GAA ATG AGA ATT ATG ACT CCA GAA GGC AAA 768 Asn Ile His Glu GluLys Glu Met Arg Ile Met Thr Pro Glu Gly Lys 245 250 255 TGG CAT GCT TCAAAA GTA TTG AGA ATT GAA AAT ATT GGT GAA AGC AAT 816 Trp His Ala Ser LysVal Leu Arg Ile Glu Asn Ile Gly Glu Ser Asn 260 265 270 CTA AAT GTT TTATAT AAT TGC ACT GTG GCC AGC ACG GGA GGC ACA GAC 864 Leu Asn Val Leu TyrAsn Cys Thr Val Ala Ser Thr Gly Gly Thr Asp 275 280 285 ACC AAA AGC TTCATC TTG GTG AGA AAA GAC ATG GCT GAT ATC CCA GGC 912 Thr Lys Ser Phe IleLeu Val Arg Lys Asp Met Ala Asp Ile Pro Gly 290 295 300 CAC GTC TTC ACAAGA 927 His Val Phe Thr Arg 305 (2) INFORMATION FOR SEQ ID NO: 7: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 1620 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: lymphoblastoid cell derivedfrom a patient with Hodgkin′s disease (C) INDIVIDUAL ISOLATE: L428 (FERMBP-5777) (ix) FEATURE: (A) NAME/KEY:sig peptide (B) LOCATION:1..57 (C)IDENTIFICATION METHOD:E (ix) FEATURE: (A) NAME/KEY:mat peptide (B)LOCATION:58..1620 (C) IDENTIFICATION METHOD:E (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 7: ATG AAT TGT AGA GAA TTA CCC TTG ACC CTT TGG GTG CTT ATATCT GTA 48 Met Asn Cys Arg Glu Leu Pro Leu Thr Leu Trp Val Leu Ile SerVal -15 -10 -5 AGC ACT GCA GAA TCT TGT ACT TCA CGT CCC CAC ATT ACT GTGGTT GAA 96 Ser Thr Ala Glu Ser Cys Thr Ser Arg Pro His Ile Thr Val ValGlu 1 5 10 GGG GAA CCT TTC TAT CTG AAA CAT TGC TCG TGT TCA CTT GCA CATGAG 144 Gly Glu Pro Phe Tyr Leu Lys His Cys Ser Cys Ser Leu Ala His Glu15 20 25 ATT GAA ACA ACC ACC AAA AGC TGG TAC AAA AGC AGT GGA TCA CAG GAA192 Ile Glu Thr Thr Thr Lys Ser Trp Tyr Lys Ser Ser Gly Ser Gln Glu 3035 40 45 CAT GTG GAG CTG AAC CCA AGG AGT TCC TCG AGA ATT GCT TTG CAT GAT240 His Val Glu Leu Asn Pro Arg Ser Ser Ser Arg Ile Ala Leu His Asp 5055 60 TGT GTT TTG GAG TTT TGG CCA GTT GAG TTG AAT GAC ACA GGA TCT TAC288 Cys Val Leu Glu Phe Trp Pro Val Glu Leu Asn Asp Thr Gly Ser Tyr 6570 75 TTT TTC CAA ATG AAA AAT TAT ACT CAG AAA TGG AAA TTA AAT GTC ATC336 Phe Phe Gln Met Lys Asn Tyr Thr Gln Lys Trp Lys Leu Asn Val Ile 8085 90 AGA AGA AAT AAA CAC AGC TGT TTC ACT GAA AGA CAA GTA ACT AGT AAA384 Arg Arg Asn Lys His Ser Cys Phe Thr Glu Arg Gln Val Thr Ser Lys 95100 105 ATT GTG GAA GTT AAA AAA TTT TTT CAG ATA ACC TGT GAA AAC AGT TAC432 Ile Val Glu Val Lys Lys Phe Phe Gln Ile Thr Cys Glu Asn Ser Tyr 110115 120 125 TAT CAA ACA CTG GTC AAC AGC ACA TCA TTG TAT AAG AAC TGT AAAAAG 480 Tyr Gln Thr Leu Val Asn Ser Thr Ser Leu Tyr Lys Asn Cys Lys Lys130 135 140 CTA CTA CTG GAG AAC AAT AAA AAC CCA ACG ATA AAG AAG AAC GCCGAG 528 Leu Leu Leu Glu Asn Asn Lys Asn Pro Thr Ile Lys Lys Asn Ala Glu145 150 155 TTT GAA GAT CAG GGG TAT TAC TCC TGC GTG CAT TTC CTT CAT CATAAT 576 Phe Glu Asp Gln Gly Tyr Tyr Ser Cys Val His Phe Leu His His Asn160 165 170 GGA AAA CTA TTT AAT ATC ACC AAA ACC TTC AAT ATA ACA ATA GTGGAA 624 Gly Lys Leu Phe Asn Ile Thr Lys Thr Phe Asn Ile Thr Ile Val Glu175 180 185 GAT CGC AGT AAT ATA GTT CCG GTT CTT CTT GGA CCA AAG CTT AACCAT 672 Asp Arg Ser Asn Ile Val Pro Val Leu Leu Gly Pro Lys Leu Asn His190 195 200 205 GTT GCA GTG GAA TTA GGA AAA AAC GTA AGG CTC AAC TGC TCTGCT TTG 720 Val Ala Val Glu Leu Gly Lys Asn Val Arg Leu Asn Cys Ser AlaLeu 210 215 220 CTG AAT GAA GAG GAT GTA ATT TAT TGG ATG TTC GGG GAA GAAAAT GGA 768 Leu Asn Glu Glu Asp Val Ile Tyr Trp Met Phe Gly Glu Glu AsnGly 225 230 235 TCG GAT CCT AAT ATA CAT GAA GAG AAA GAA ATG AGA ATT ATGACT CCA 816 Ser Asp Pro Asn Ile His Glu Glu Lys Glu Met Arg Ile Met ThrPro 240 245 250 GAA GGC AAA TGG CAT GCT TCA AAA GTA TTG AGA ATT GAA AATATT GGT 864 Glu Gly Lys Trp His Ala Ser Lys Val Leu Arg Ile Glu Asn IleGly 255 260 265 GAA AGC AAT CTA AAT GTT TTA TAT AAT TGC ACT GTG GCC AGCACG GGA 912 Glu Ser Asn Leu Asn Val Leu Tyr Asn Cys Thr Val Ala Ser ThrGly 270 275 280 285 GGC ACA GAC ACC AAA AGC TTC ATC TTG GTG AGA AAA GACATG GCT GAT 960 Gly Thr Asp Thr Lys Ser Phe Ile Leu Val Arg Lys Asp MetAla Asp 290 295 300 ATC CCA GGC CAC GTC TTC ACA AGA GGA ATG ATC ATA GCTGTT TTG ATC 1008 Ile Pro Gly His Val Phe Thr Arg Gly Met Ile Ile Ala ValLeu Ile 305 310 315 TTG GTG GCA GTA GTG TGC CTA GTG ACT GTG TGT GTC ATTTAT AGA GTT 1056 Leu Val Ala Val Val Cys Leu Val Thr Val Cys Val Ile TyrArg Val 320 325 330 GAC TTG GTT CTA TTT TAT AGA CAT TTA ACG AGA AGA GATGAA ACA TTA 1104 Asp Leu Val Leu Phe Tyr Arg His Leu Thr Arg Arg Asp GluThr Leu 335 340 345 ACA GAT GGA AAA ACA TAT GAT GCT TTT GTG TCT TAC CTAAAA GAA TGC 1152 Thr Asp Gly Lys Thr Tyr Asp Ala Phe Val Ser Tyr Leu LysGlu Cys 350 355 360 365 CGA CCT GAA AAT GGA GAG GAG CAC ACC TTT GCT GTGGAG ATT TTG CCC 1200 Arg Pro Glu Asn Gly Glu Glu His Thr Phe Ala Val GluIle Leu Pro 370 375 380 AGG GTG TTG GAG AAA CAT TTT GGG TAT AAG TTA TGCATA TTT GAA AGG 1248 Arg Val Leu Glu Lys His Phe Gly Tyr Lys Leu Cys IlePhe Glu Arg 385 390 395 GAT GTA GTG CCT GGA GGA GCT GTT GTT GAT GAA ATCCAC TCA CTG ATA 1296 Asp Val Val Pro Gly Gly Ala Val Val Asp Glu Ile HisSer Leu Ile 400 405 410 GAG AAA AGC CGA AGA CTA ATC ATT GTC CTA AGT AAAAGT TAT ATG TCT 1344 Glu Lys Ser Arg Arg Leu Ile Ile Val Leu Ser Lys SerTyr Met Ser 415 420 425 AAT GAG GTC AGG TAT GAA CTT GAA AGT GGA CTC CATGAA GCA TTG GTG 1392 Asn Glu Val Arg Tyr Glu Leu Glu Ser Gly Leu His GluAla Leu Val 430 435 440 445 GAA AGA AAA ATT AAA ATA ATC TTA ATT GAA TTTACA CCT GTT ACT GAC 1440 Glu Arg Lys Ile Lys Ile Ile Leu Ile Glu Phe ThrPro Val Thr Asp 450 455 460 TTC ACA TTC TTG CCC CAA TCA CTA AAG CTT TTGAAA TCT CAC AGA GTT 1488 Phe Thr Phe Leu Pro Gln Ser Leu Lys Leu Leu LysSer His Arg Val 465 470 475 CTG AAG TGG AAG GCC GAT AAA TCT CTT TCT TATAAC TCA AGG TTC TGG 1536 Leu Lys Trp Lys Ala Asp Lys Ser Leu Ser Tyr AsnSer Arg Phe Trp 480 485 490 AAG AAC CTT CTT TAC TTA ATG CCT GCA AAA ACAGTC AAG CCA GGT AGA 1584 Lys Asn Leu Leu Tyr Leu Met Pro Ala Lys Thr ValLys Pro Gly Arg 495 500 505 GAC GAA CCG GAA GTC TTG CCT GTT CTT TCC GAGTCT 1620 Asp Glu Pro Glu Val Leu Pro Val Leu Ser Glu Ser 510 515 520 (2)INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:369 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY:sigpeptide (B) LOCATION:1..57 (C) IDENTIFICATION METHOD:S (ix) FEATURE: (A)NAME/KEY:mat peptide (B) LOCATION:58..369 (C) IDENTIFICATION METHOD:S(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: ATG AAT TGT AGA GAA TTA CCC TTGACC CTT TGG GTG CTT ATA TCT GTA 48 Met Asn Cys Arg Glu Leu Pro Leu ThrLeu Trp Val Leu Ile Ser Val -15 -10 -5 AGC ACT GCA GAA TCT TGT ACT TCACGT CCC CAC ATT ACT GTG GTT GAA 96 Ser Thr Ala Glu Ser Cys Thr Ser ArgPro His Ile Thr Val Val Glu 1 5 10 GGG GAA CCT TTC TAT CTG AAA CAT TGCTCG TGT TCA CTT GCA CAT GAG 144 Gly Glu Pro Phe Tyr Leu Lys His Cys SerCys Ser Leu Ala His Glu 15 20 25 ATT GAA ACA ACC ACC AAA AGC TGG TAC AAAAGC AGT GGA TCA CAG GAA 192 Ile Glu Thr Thr Thr Lys Ser Trp Tyr Lys SerSer Gly Ser Gln Glu 30 35 40 45 CAT GTG GAG CTG AAC CCA AGG AGT TCC TCGAGA ATT GCT TTG CAT GAT 240 His Val Glu Leu Asn Pro Arg Ser Ser Ser ArgIle Ala Leu His Asp 50 55 60 TGT GTT TTG GAG TTT TGG CCA GTT GAG TTG AATGAC ACA GGA TCT TAC 288 Cys Val Leu Glu Phe Trp Pro Val Glu Leu Asn AspThr Gly Ser Tyr 65 70 75 TTT TTC CAA ATG AAA AAT TAT ACT CAG AAA TGG AAATTA AAT GTC ATC 336 Phe Phe Gln Met Lys Asn Tyr Thr Gln Lys Trp Lys LeuAsn Val Ile 80 85 90 AGA AGA AAT AAA CAC AGC TGT TTC ACT GAA AGA 369 ArgArg Asn Lys His Ser Cys Phe Thr Glu Arg 95 100 (2) INFORMATION FOR SEQID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 678 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii)MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY:sig peptide (B)LOCATION:1..57 (C) IDENTIFICATION METHOD:S (ix) FEATURE: (A)NAME/KEY:mat peptide (B) LOCATION:58..678 (C) IDENTIFICATION METHOD:S(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: ATG AAT TGT AGA GAA TTA CCC TTGACC CTT TGG GTG CTT ATA TCT GTA 48 Met Asn Cys Arg Glu Leu Pro Leu ThrLeu Trp Val Leu Ile Ser Val -15 -10 -5 AGC ACT GCA GAA TCT TGT ACT TCACGT CCC CAC ATT ACT GTG GTT GAA 96 Ser Thr Ala Glu Ser Cys Thr Ser ArgPro His Ile Thr Val Val Glu 1 5 10 GGG GAA CCT TTC TAT CTG AAA CAT TGCTCG TGT TCA CTT GCA CAT GAG 144 Gly Glu Pro Phe Tyr Leu Lys His Cys SerCys Ser Leu Ala His Glu 15 20 25 ATT GAA ACA ACC ACC AAA AGC TGG TAC AAAAGC AGT GGA TCA CAG GAA 192 Ile Glu Thr Thr Thr Lys Ser Trp Tyr Lys SerSer Gly Ser Gln Glu 30 35 40 45 CAT GTG GAG CTG AAC CCA AGG AGT TCC TCGAGA ATT GCT TTG CAT GAT 240 His Val Glu Leu Asn Pro Arg Ser Ser Ser ArgIle Ala Leu His Asp 50 55 60 TGT GTT TTG GAG TTT TGG CCA GTT GAG TTG AATGAC ACA GGA TCT TAC 288 Cys Val Leu Glu Phe Trp Pro Val Glu Leu Asn AspThr Gly Ser Tyr 65 70 75 TTT TTC CAA ATG AAA AAT TAT ACT CAG AAA TGG AAATTA AAT GTC ATC 336 Phe Phe Gln Met Lys Asn Tyr Thr Gln Lys Trp Lys LeuAsn Val Ile 80 85 90 AGA AGA AAT AAA CAC AGC TGT TTC ACT GAA AGA CAA GTAACT AGT AAA 384 Arg Arg Asn Lys His Ser Cys Phe Thr Glu Arg Gln Val ThrSer Lys 95 100 105 ATT GTG GAA GTT AAA AAA TTT TTT CAG ATA ACC TGT GAAAAC AGT TAC 432 Ile Val Glu Val Lys Lys Phe Phe Gln Ile Thr Cys Glu AsnSer Tyr 110 115 120 125 TAT CAA ACA CTG GTC AAC AGC ACA TCA TTG TAT AAGAAC TGT AAA AAG 480 Tyr Gln Thr Leu Val Asn Ser Thr Ser Leu Tyr Lys AsnCys Lys Lys 130 135 140 CTA CTA CTG GAG AAC AAT AAA AAC CCA ACG ATA AAGAAG AAC GCC GAG 528 Leu Leu Leu Glu Asn Asn Lys Asn Pro Thr Ile Lys LysAsn Ala Glu 145 150 155 TTT GAA GAT CAG GGG TAT TAC TCC TGC GTG CAT TTCCTT CAT CAT AAT 576 Phe Glu Asp Gln Gly Tyr Tyr Ser Cys Val His Phe LeuHis His Asn 160 165 170 GGA AAA CTA TTT AAT ATC ACC AAA ACC TTC AAT ATAACA ATA GTG GAA 624 Gly Lys Leu Phe Asn Ile Thr Lys Thr Phe Asn Ile ThrIle Val Glu 175 180 185 GAT CGC AGT AAT ATA GTT CCG GTT CTT CTT GGA CCAAAG CTT AAC CAT 672 Asp Arg Ser Asn Ile Val Pro Val Leu Leu Gly Pro LysLeu Asn His 190 195 200 205 GTT GCA 678 Val Ala (2) INFORMATION FOR SEQID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 984 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii)MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY:sig peptide (B)LOCATION:1..57 (C) IDENTIFICATION METHOD:E (ix) FEATURE: (A)NAME/KEY:mat peptide (B) LOCATION:58..984 (C) IDENTIFICATION METHOD:E(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: ATG AAT TGT AGA GAA TTA CCCTTG ACC CTT TGG GTG CTT ATA TCT GTA 48 Met Asn Cys Arg Glu Leu Pro LeuThr Leu Trp Val Leu Ile Ser Val -15 -10 -5 AGC ACT GCA GAA TCT TGT ACTTCA CGT CCC CAC ATT ACT GTG GTT GAA 96 Ser Thr Ala Glu Ser Cys Thr SerArg Pro His Ile Thr Val Val Glu 1 5 10 GGG GAA CCT TTC TAT CTG AAA CATTGC TCG TGT TCA CTT GCA CAT GAG 144 Gly Glu Pro Phe Tyr Leu Lys His CysSer Cys Ser Leu Ala His Glu 15 20 25 ATT GAA ACA ACC ACC AAA AGC TGG TACAAA AGC AGT GGA TCA CAG GAA 192 Ile Glu Thr Thr Thr Lys Ser Trp Tyr LysSer Ser Gly Ser Gln Glu 30 35 40 45 CAT GTG GAG CTG AAC CCA AGG AGT TCCTCG AGA ATT GCT TTG CAT GAT 240 His Val Glu Leu Asn Pro Arg Ser Ser SerArg Ile Ala Leu His Asp 50 55 60 TGT GTT TTG GAG TTT TGG CCA GTT GAG TTGAAT GAC ACA GGA TCT TAC 288 Cys Val Leu Glu Phe Trp Pro Val Glu Leu AsnAsp Thr Gly Ser Tyr 65 70 75 TTT TTC CAA ATG AAA AAT TAT ACT CAG AAA TGGAAA TTA AAT GTC ATC 336 Phe Phe Gln Met Lys Asn Tyr Thr Gln Lys Trp LysLeu Asn Val Ile 80 85 90 AGA AGA AAT AAA CAC AGC TGT TTC ACT GAA AGA CAAGTA ACT AGT AAA 384 Arg Arg Asn Lys His Ser Cys Phe Thr Glu Arg Gln ValThr Ser Lys 95 100 105 ATT GTG GAA GTT AAA AAA TTT TTT CAG ATA ACC TGTGAA AAC AGT TAC 432 Ile Val Glu Val Lys Lys Phe Phe Gln Ile Thr Cys GluAsn Ser Tyr 110 115 120 125 TAT CAA ACA CTG GTC AAC AGC ACA TCA TTG TATAAG AAC TGT AAA AAG 480 Tyr Gln Thr Leu Val Asn Ser Thr Ser Leu Tyr LysAsn Cys Lys Lys 130 135 140 CTA CTA CTG GAG AAC AAT AAA AAC CCA ACG ATAAAG AAG AAC GCC GAG 528 Leu Leu Leu Glu Asn Asn Lys Asn Pro Thr Ile LysLys Asn Ala Glu 145 150 155 TTT GAA GAT CAG GGG TAT TAC TCC TGC GTG CATTTC CTT CAT CAT AAT 576 Phe Glu Asp Gln Gly Tyr Tyr Ser Cys Val His PheLeu His His Asn 160 165 170 GGA AAA CTA TTT AAT ATC ACC AAA ACC TTC AATATA ACA ATA GTG GAA 624 Gly Lys Leu Phe Asn Ile Thr Lys Thr Phe Asn IleThr Ile Val Glu 175 180 185 GAT CGC AGT AAT ATA GTT CCG GTT CTT CTT GGACCA AAG CTT AAC CAT 672 Asp Arg Ser Asn Ile Val Pro Val Leu Leu Gly ProLys Leu Asn His 190 195 200 205 GTT GCA GTG GAA TTA GGA AAA AAC GTA AGGCTC AAC TGC TCT GCT TTG 720 Val Ala Val Glu Leu Gly Lys Asn Val Arg LeuAsn Cys Ser Ala Leu 210 215 220 CTG AAT GAA GAG GAT GTA ATT TAT TGG ATGTTC GGG GAA GAA AAT GGA 768 Leu Asn Glu Glu Asp Val Ile Tyr Trp Met PheGly Glu Glu Asn Gly 225 230 235 TCG GAT CCT AAT ATA CAT GAA GAG AAA GAAATG AGA ATT ATG ACT CCA 816 Ser Asp Pro Asn Ile His Glu Glu Lys Glu MetArg Ile Met Thr Pro 240 245 250 GAA GGC AAA TGG CAT GCT TCA AAA GTA TTGAGA ATT GAA AAT ATT GGT 864 Glu Gly Lys Trp His Ala Ser Lys Val Leu ArgIle Glu Asn Ile Gly 255 260 265 GAA AGC AAT CTA AAT GTT TTA TAT AAT TGCACT GTG GCC AGC ACG GGA 912 Glu Ser Asn Leu Asn Val Leu Tyr Asn Cys ThrVal Ala Ser Thr Gly 270 275 280 285 GGC ACA GAC ACC AAA AGC TTC ATC TTGGTG AGA AAA GAC ATG GCT GAT 960 Gly Thr Asp Thr Lys Ser Phe Ile Leu ValArg Lys Asp Met Ala Asp 290 295 300 ATC CCA GGC CAC GTC TTC ACA AGA 984Ile Pro Gly His Val Phe Thr Arg 305 (2) INFORMATION FOR SEQ ID NO: 11:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 975 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULETYPE: cDNA (ix) FEATURE: (A) NAME/KEY:sig peptide (B) LOCATION:1..54 (C)IDENTIFICATION METHOD:S (ix) FEATURE: (A) NAME/KEY:mat peptide (B)LOCATION:55..975 (C) IDENTIFICATION METHOD:S (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 11: ATG CAT CAT GAA GAA TTA ATC TTG ACA CTC TGC ATT CTC ATTGTT AAA 48 Met His His Glu Glu Leu Ile Leu Thr Leu Cys Ile Leu Ile ValLys -15 -10 -5 AGT GCC TCA AAA AGT TGT ATT CAC CGA TCA CAA ATT CAT GTGGTA GAG 96 Ser Ala Ser Lys Ser Cys Ile His Arg Ser Gln Ile His Val ValGlu 1 5 10 GGA GAA CCT TTT TAT CTG AAG CCA TGT GGC ATA TCT GCA CCA GTGCAC 144 Gly Glu Pro Phe Tyr Leu Lys Pro Cys Gly Ile Ser Ala Pro Val His15 20 25 30 AGG AAT GAA ACA GCC ACC ATG AGA TGG TTC AAA GGC AGT GCT TCACAT 192 Arg Asn Glu Thr Ala Thr Met Arg Trp Phe Lys Gly Ser Ala Ser His35 40 45 GAG TAT AGA GAG CTG AAC AAC AGA AGC TCG CCC AGA GTC ACT TTT CAT240 Glu Tyr Arg Glu Leu Asn Asn Arg Ser Ser Pro Arg Val Thr Phe His 5055 60 GAT CAC ACC TTG GAA TTC TGG CCA GTT GAG ATG GAG GAT GAG GGA ACG288 Asp His Thr Leu Glu Phe Trp Pro Val Glu Met Glu Asp Glu Gly Thr 6570 75 TAC ATT TCT CAA GTC GGA AAT GAT CGT CGC AAT TGG ACC TTA AAT GTC336 Tyr Ile Ser Gln Val Gly Asn Asp Arg Arg Asn Trp Thr Leu Asn Val 8085 90 ACC AAA AGA AAC AAA CAC AGC TGT TTC TCT GAC AAG CTC GTG ACA AGC384 Thr Lys Arg Asn Lys His Ser Cys Phe Ser Asp Lys Leu Val Thr Ser 95100 105 110 AGA GAT GTT GAA GTT AAC AAA TCT CTG CAT ATC ACT TGT AAG AATCCT 432 Arg Asp Val Glu Val Asn Lys Ser Leu His Ile Thr Cys Lys Asn Pro115 120 125 AAC TAT GAA GAG CTG ATC CAG GAC ACA TGG CTG TAT AAG AAC TGTAAG 480 Asn Tyr Glu Glu Leu Ile Gln Asp Thr Trp Leu Tyr Lys Asn Cys Lys130 135 140 GAA ATA TCC AAA ACC CCA AGG ATC CTG AAG GAT GCC GAG TTT GGAGAT 528 Glu Ile Ser Lys Thr Pro Arg Ile Leu Lys Asp Ala Glu Phe Gly Asp145 150 155 GAG GGC TAC TAC TCC TGC GTG TTT TCT GTC CAC CAT AAT GGG ACACGG 576 Glu Gly Tyr Tyr Ser Cys Val Phe Ser Val His His Asn Gly Thr Arg160 165 170 TAC AAC ATC ACC AAG ACT GTC AAT ATA ACA GTT ATT GAA GGA AGGAGT 624 Tyr Asn Ile Thr Lys Thr Val Asn Ile Thr Val Ile Glu Gly Arg Ser175 180 185 190 AAA GTA ACT CCA GCT ATT TTA GGA CCA AAG TGT GAG AAG GTTGGT GTA 672 Lys Val Thr Pro Ala Ile Leu Gly Pro Lys Cys Glu Lys Val GlyVal 195 200 205 GAA CTA GGA AAG GAT GTG GAG TTG AAC TGC AGT GCT TCA TTGAAT AAA 720 Glu Leu Gly Lys Asp Val Glu Leu Asn Cys Ser Ala Ser Leu AsnLys 210 215 220 GAC GAT CTG TTT TAT TGG AGC ATC AGG AAA GAG GAC AGC TCAGAC CCT 768 Asp Asp Leu Phe Tyr Trp Ser Ile Arg Lys Glu Asp Ser Ser AspPro 225 230 235 AAT GTG CAA GAA GAC AGG AAG GAG ACG ACA ACA TGG ATT TCTGAA GGC 816 Asn Val Gln Glu Asp Arg Lys Glu Thr Thr Thr Trp Ile Ser GluGly 240 245 250 AAA CTG CAT GCT TCA AAA ATA CTG AGA TTT CAG AAA ATT ACTGAA AAC 864 Lys Leu His Ala Ser Lys Ile Leu Arg Phe Gln Lys Ile Thr GluAsn 255 260 265 270 TAT CTC AAT GTT TTA TAT AAT TGC ACC GTG GCC AAC GAAGAA GCC ATA 912 Tyr Leu Asn Val Leu Tyr Asn Cys Thr Val Ala Asn Glu GluAla Ile 275 280 285 GAC ACC AAG AGC TTC GTC TTG GTG AGA AAA GAA ATA CCTGAT ATC CCA 960 Asp Thr Lys Ser Phe Val Leu Val Arg Lys Glu Ile Pro AspIle Pro 290 295 300 GGC CAT GTC TTT ACA 975 Gly His Val Phe Thr 305 (2)INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:5 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(v) FRAGMENT TYPE: internal fragment (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 12: Trp His Ala Ser Lys 1 (2) INFORMATION FOR SEQ ID NO: 13: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internalfragment (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: Ile Met Thr Pro GluGly Lys 1 5 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 13 (B) TYPE: amino acid (D) TOPOLOGY:linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal fragment(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: Ser Ser Gly Ser Gln Glu HisVal Glu Leu Asn Pro Arg 1 5 10 (2) INFORMATION FOR SEQ ID NO: 15: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internalfragment (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: Ser Trp Tyr Lys 1 (2)INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:10 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(v) FRAGMENT TYPE: internal fragment (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 16: Leu Asn His Val Ala Val Glu Leu Gly Lys 1 5 10 (2) INFORMATIONFOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 (B) TYPE:amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENTTYPE: internal fragment (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: SerPhe Ile Leu Val Arg 1 5 (2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 15 (B) TYPE: amino acid (D) TOPOLOGY:linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal fragment(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: Thr Val Lys Pro Gly Arg AspGlu Pro Glu Val Leu Pro Val Leu 1 5 10 15 (2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 (B) TYPE: amino acid(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE:internal fragment (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: Ser Asn IleVal Pro Val Leu Leu Gly Pro Lys 1 5 10 (2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 521 (B) TYPE: amino acid(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 20: Glu Ser Cys Thr Ser Arg Pro His Ile Thr ValVal Glu Gly Glu Pro 1 5 10 15 Phe Tyr Leu Lys His Cys Ser Cys Ser LeuAla His Glu Ile Glu Thr 20 25 30 Thr Thr Lys Ser Trp Tyr Lys Ser Ser GlySer Gln Glu His Val Glu 35 40 45 Leu Asn Pro Arg Ser Ser Ser Arg Ile AlaLeu His Asp Cys Val Leu 50 55 60 Glu Phe Trp Pro Val Glu Leu Asn Asp ThrGly Ser Tyr Phe Phe Gln 65 70 75 80 Met Lys Asn Tyr Thr Gln Lys Trp LysLeu Asn Val Ile Arg Arg Asn 85 90 95 Lys His Ser Cys Phe Thr Glu Arg GlnVal Thr Ser Lys Ile Val Glu 100 105 110 Val Lys Lys Phe Phe Gln Ile ThrCys Glu Asn Ser Tyr Tyr Gln Thr 115 120 125 Leu Val Asn Ser Thr Ser LeuTyr Lys Asn Cys Lys Lys Leu Leu Leu 130 135 140 Glu Asn Asn Lys Asn ProThr Ile Lys Lys Asn Ala Glu Phe Glu Asp 145 150 155 160 Gln Gly Tyr TyrSer Cys Val His Phe Leu His His Asn Gly Lys Leu 165 170 175 Phe Asn IleThr Lys Thr Phe Asn Ile Thr Ile Val Glu Asp Arg Ser 180 185 190 Asn IleVal Pro Val Leu Leu Gly Pro Lys Leu Asn His Val Ala Val 195 200 205 GluLeu Gly Lys Asn Val Arg Leu Asn Cys Ser Ala Leu Leu Asn Glu 210 215 220Glu Asp Val Ile Tyr Trp Met Phe Gly Glu Glu Asn Gly Ser Asp Pro 225 230235 240 Asn Ile His Glu Glu Lys Glu Met Arg Ile Met Thr Pro Glu Gly Lys245 250 255 Trp His Ala Ser Lys Val Leu Arg Ile Glu Asn Ile Gly Glu SerAsn 260 265 270 Leu Asn Val Leu Tyr Asn Cys Thr Val Ala Ser Thr Gly GlyThr Asp 275 280 285 Thr Lys Ser Phe Ile Leu Val Arg Lys Asp Met Ala AspIle Pro Gly 290 295 300 His Val Phe Thr Arg Gly Met Ile Ile Ala Val LeuIle Leu Val Ala 305 310 315 320 Val Val Cys Leu Val Thr Val Cys Val IleTyr Arg Val Asp Leu Val 325 330 335 Leu Phe Tyr Arg His Leu Thr Arg ArgAsp Glu Thr Leu Thr Asp Gly 340 345 350 Lys Thr Tyr Asp Ala Phe Val SerTyr Leu Lys Glu Cys Arg Pro Glu 355 360 365 Asn Gly Glu Glu His Thr PheAla Val Glu Ile Leu Pro Arg Val Leu 370 375 380 Glu Lys His Phe Gly TyrLys Leu Cys Ile Phe Glu Arg Asp Val Val 385 390 395 400 Pro Gly Gly AlaVal Val Asp Glu Ile His Ser Leu Ile Glu Lys Ser 405 410 415 Arg Arg LeuIle Ile Val Leu Ser Lys Ser Tyr Met Ser Asn Glu Val 420 425 430 Arg TyrGlu Leu Glu Ser Gly Leu His Glu Ala Leu Val Glu Arg Lys 435 440 445 IleLys Ile Ile Leu Ile Glu Phe Thr Pro Val Thr Asp Phe Thr Phe 450 455 460Leu Pro Gln Ser Leu Lys Leu Leu Lys Ser His Arg Val Leu Lys Trp 465 470475 480 Lys Ala Asp Lys Ser Leu Ser Tyr Asn Ser Arg Phe Trp Lys Asn Leu485 490 495 Leu Tyr Leu Met Pro Ala Lys Thr Val Lys Pro Gly Arg Asp GluPro 500 505 510 Glu Val Leu Pro Val Leu Ser Glu Ser 515 520 (2)INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:519 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE:peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: Ser Lys Ser Cys IleHis Arg Ser Gln Ile His Val Val Glu Gly Glu 1 5 10 15 Pro Phe Tyr LeuLys Pro Cys Gly Ile Ser Ala Pro Val His Arg Asn 20 25 30 Glu Thr Ala ThrMet Arg Trp Phe Lys Gly Ser Ala Ser His Glu Tyr 35 40 45 Arg Glu Leu AsnAsn Arg Ser Ser Pro Arg Val Thr Phe His Asp His 50 55 60 Thr Leu Glu PheTrp Pro Val Glu Met Glu Asp Glu Gly Thr Tyr Ile 65 70 75 80 Ser Gln ValGly Asn Asp Arg Arg Asn Trp Thr Leu Asn Val Thr Lys 85 90 95 Arg Asn LysHis Ser Cys Phe Ser Asp Lys Leu Val Thr Ser Arg Asp 100 105 110 Val GluVal Asn Lys Ser Leu His Ile Thr Cys Lys Asn Pro Asn Tyr 115 120 125 GluGlu Leu Ile Gln Asp Thr Trp Leu Tyr Lys Asn Cys Lys Glu Ile 130 135 140Ser Lys Thr Pro Arg Ile Leu Lys Asp Ala Glu Phe Gly Asp Glu Gly 145 150155 160 Tyr Tyr Ser Cys Val Phe Ser Val His His Asn Gly Thr Arg Tyr Asn165 170 175 Ile Thr Lys Thr Val Asn Ile Thr Val Ile Glu Gly Arg Ser LysVal 180 185 190 Thr Pro Ala Ile Leu Gly Pro Lys Cys Glu Lys Val Gly ValGlu Leu 195 200 205 Gly Lys Asp Val Glu Leu Asn Cys Ser Ala Ser Leu AsnLys Asp Asp 210 215 220 Leu Phe Tyr Trp Ser Ile Arg Lys Glu Asp Ser SerAsp Pro Asn Val 225 230 235 240 Gln Glu Asp Arg Lys Glu Thr Thr Thr TrpIle Ser Glu Gly Lys Leu 245 250 255 His Ala Ser Lys Ile Leu Arg Phe GlnLys Ile Thr Glu Asn Tyr Leu 260 265 270 Asn Val leu Tyr Asn Cys Thr ValAla Asn Glu Glu Ala Ile Asp Thr 275 280 285 Lys Ser Phe Val Leu Val ArgLys Glu Ile Pro Asp Ile Pro Gly His 290 295 300 Val Phe Thr Gly Gly ValThr Val Leu Val Leu Ala Ser Val Ala Ala 305 310 315 320 Val Cys Ile ValIle Leu Cys Val Ile Tyr Lys Val Asp Leu Val Leu 325 330 335 Phe Tyr ArgArg Ile Ala Glu Arg Asp Glu Thr Leu Thr Asp Gly Lys 340 345 350 Thr TyrAsp Ala Phe Val Ser Tyr Leu Lys Glu Cys His Pro Glu Asn 355 360 365 LysGlu Glu Tyr Thr Phe Ala Val Glu Thr Leu Pro Arg Val Leu Glu 370 375 380Lys Gln Phe Gly Tyr Lys Leu Cys Ile Phe Glu Arg Asp Val Val Pro 385 390395 400 Gly Gly Ala Val Val Glu Glu Ile His Ser Leu Ile Glu Lys Ser Arg405 410 415 Arg Leu Ile Ile Val Leu Ser Gln Ser Tyr Leu Thr Asn Gly AlaArg 420 425 430 Arg Glu Leu Glu Ser Gly Leu His Glu Ala Leu Val Glu ArgLys Ile 435 440 445 Lys Ile Ile Leu Ile Glu Phe Thr Pro Ala Ser Asn IleThr Phe Leu 450 455 460 Pro Pro Ser Leu Lys Leu Leu Lys Ser Tyr Arg ValLeu Lys Trp Arg 465 470 475 480 Ala Asp Ser Pro Ser Met Asn Ser Arg PheTrp Lys Asn Leu Val Tyr 485 490 495 Leu Met Pro Ala Lys Ala Val Lys ProTrp Arg Glu Glu Ser Glu Ala 500 505 510 Arg Ser Val Leu Ser Ala Pro 515(2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 309 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULETYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: Glu Ser Cys ThrSer Arg Pro His Ile Thr Val Val Glu Gly Glu Pro 1 5 10 15 Phe Tyr LeuLys His Cys Ser Cys Ser Leu Ala His Glu Ile Glu Thr 20 25 30 Thr Thr LysSer Trp Tyr Lys Ser Ser Gly Ser Gln Glu His Val Glu 35 40 45 Leu Asn ProArg Ser Ser Ser Arg Ile Ala Leu His Asp Cys Val Leu 50 55 60 Glu Phe TrpPro Val Glu Leu Asn Asp Thr Gly Ser Tyr Phe Phe Gln 65 70 75 80 Met LysAsn Tyr Thr Gln Lys Trp Lys Leu Asn Val Ile Arg Arg Asn 85 90 95 Lys HisSer Cys Phe Thr Glu Arg Gln Val Thr Ser Lys Ile Val Glu 100 105 110 ValLys Lys Phe Phe Gln Ile Thr Cys Glu Asn Ser Tyr Tyr Gln Thr 115 120 125Leu Val Asn Ser Thr Ser Leu Tyr Lys Asn Cys Lys Lys Leu Leu Leu 130 135140 Glu Asn Asn Lys Asn Pro Thr Ile Lys Lys Asn Ala Glu Phe Glu Asp 145150 155 160 Gln Gly Tyr Tyr Ser Cys Val His Phe Leu His His Asn Gly LysLeu 165 170 175 Phe Asn Ile Thr Lys Thr Phe Asn Ile Thr Ile Val Glu AspArg Ser 180 185 190 Asn Ile Val Pro Val Leu Leu Gly Pro Lys Leu Asn HisVal Ala Val 195 200 205 Glu Leu Gly Lys Asn Val Arg Leu Asn Cys Ser AlaLeu Leu Asn Glu 210 215 220 Glu Asp Val Ile Tyr Trp Met Phe Gly Glu GluAsn Gly Ser Asp Pro 225 230 235 240 Asn Ile His Glu Glu Lys Glu Met ArgIle Met Thr Pro Glu Gly Lys 245 250 255 Trp His Ala Ser Lys Val Leu ArgIle Glu Asn Ile Gly Glu Ser Asn 260 265 270 Leu Asn Val Leu Tyr Asn CysThr Val Ala Ser Thr Gly Gly Thr Asp 275 280 285 Thr Lys Ser Phe Ile LeuVal Arg Lys Asp Met Ala Asp Ile Pro Gly 290 295 300 His Val Phe Thr Arg305 (2) INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 207 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULETYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: Glu Ser Cys ThrSer Arg Pro His Ile Thr Val Val Glu Gly Glu Pro 1 5 10 15 Phe Tyr LeuLys His Cys Ser Cys Ser Leu Ala His Glu Ile Glu Thr 20 25 30 Thr Thr LysSer Trp Tyr Lys Ser Ser Gly Ser Gln Glu His Val Glu 35 40 45 Leu Asn ProArg Ser Ser Ser Arg Ile Ala Leu His Asp Cys Val Leu 50 55 60 Glu Phe TrpPro Val Glu Leu Asn Asp Thr Gly Ser Tyr Phe Phe Gln 65 70 75 80 Met LysAsn Tyr Thr Gln Lys Trp Lys Leu Asn Val Ile Arg Arg Asn 85 90 95 Lys HisSer Cys Phe Thr Glu Arg Gln Val Thr Ser Lys Ile Val Glu 100 105 110 ValLys Lys Phe Phe Gln Ile Thr Cys Glu Asn Ser Tyr Tyr Gln Thr 115 120 125Leu Val Asn Ser Thr Ser Leu Tyr Lys Asn Cys Lys Lys Leu Leu Leu 130 135140 Glu Asn Asn Lys Asn Pro Thr Ile Lys Lys Asn Ala Glu Phe Glu Asp 145150 155 160 Gln Gly Tyr Tyr Ser Cys Val His Phe Leu His His Asn Gly LysLeu 165 170 175 Phe Asn Ile Thr Lys Thr Phe Asn Ile Thr Ile Val Glu AspArg Ser 180 185 190 Asn Ile Val Pro Val Leu Leu Gly Pro Lys Leu Asn HisVal Ala 195 200 205 (2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 104 (B) TYPE: amino acid (D) TOPOLOGY:linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: Glu Ser Cys Thr Ser Arg Pro His Ile Thr Val Val Glu Gly Glu Pro 1 510 15 Phe Tyr Leu Lys His Cys Ser Cys Ser Leu Ala His Glu Ile Glu Thr 2025 30 Thr Thr Lys Ser Trp Tyr Lys Ser Ser Gly Ser Gln Glu His Val Glu 3540 45 Leu Asn Pro Arg Ser Ser Ser Arg Ile Ala Leu His Asp Cys Val Leu 5055 60 Glu Phe Trp Pro Val Glu Leu Asn Asp Thr Gly Ser Tyr Phe Phe Gln 6570 75 80 Met Lys Asn Tyr Thr Gln Lys Trp Lys Leu Asn Val Ile Arg Arg Asn85 90 95 Lys His Ser Cys Phe Thr Glu Arg 100 (2) INFORMATION FOR SEQ IDNO: 25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 307 (B) TYPE: aminoacid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 25: Ser Lys Ser Cys Ile His Arg Ser Gln Ile HisVal Val Glu Gly Glu 1 5 10 15 Pro Phe Tyr Leu Lys Pro Cys Gly Ile SerAla Pro Val His Arg Asn 20 25 30 Glu Thr Ala Thr Met Arg Trp Phe Lys GlySer Ala Ser His Glu Tyr 35 40 45 Arg Glu Leu Asn Asn Arg Ser Ser Pro ArgVal Thr Phe His Asp His 50 55 60 Thr Leu Glu Phe Trp Pro Val Glu Met GluAsp Glu Gly Thr Tyr Ile 65 70 75 80 Ser Gln Val Gly Asn Asp Arg Arg AsnTrp Thr Leu Asn Val Thr Lys 85 90 95 Arg Asn Lys His Ser Cys Phe Ser AspLys Leu Val Thr Ser Arg Asp 100 105 110 Val Glu Val Asn Lys Ser Leu HisIle Thr Cys Lys Asn Pro Asn Tyr 115 120 125 Glu Glu Leu Ile Gln Asp ThrTrp Leu Tyr Lys Asn Cys Lys Glu Ile 130 135 140 Ser Lys Thr Pro Arg IleLeu Lys Asp Ala Glu Phe Gly Asp Glu Gly 145 150 155 160 Tyr Tyr Ser CysVal Phe Ser Val His His Asn Gly Thr Arg Tyr Asn 165 170 175 Ile Thr LysThr Val Asn Ile Thr Val Ile Glu Gly Arg Ser Lys Val 180 185 190 Thr ProAla Ile Leu Gly Pro Lys Cys Glu Lys Val Gly Val Glu Leu 195 200 205 GlyLys Asp Val Glu Leu Asn Cys Ser Ala Ser Leu Asn Lys Asp Asp 210 215 220Leu Phe Tyr Trp Ser Ile Arg Lys Glu Asp Ser Ser Asp Pro Asn Val 225 230235 240 Gln Glu Asp Arg Lys Glu Thr Thr Thr Trp Ile Ser Glu Gly Lys Leu245 250 255 His Ala Ser Lys Ile Leu Arg Phe Gln Lys Ile Thr Glu Asn TyrLeu 260 265 270 Asn Val Leu Tyr Asn Cys Thr Val Ala Asn Glu Glu Ala IleAsp Thr 275 280 285 Lys Ser Phe Val Leu Val Arg Lys Glu Ile Pro Asp IlePro Gly His 290 295 300 Val Phe Thr 305 (2) INFORMATION FOR SEQ ID NO:26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 157 (B) TYPE: amino acid(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 26: Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser ValIle Arg Asn Leu Asn 1 5 10 15 Asp Gln Val Leu Phe Ile Asp Gln Gly AsnArg Pro Leu Phe Glu Asp 20 25 30 Met Thr Asp Ser Asp Cys Arg Asp Asn AlaPro Arg Thr Ile Phe Ile 35 40 45 Ile Ser Met Tyr Lys Asp Ser Gln Pro ArgGly Met Ala Val Thr Ile 50 55 60 Ser Val Lys Cys Glu Lys Ile Ser Xaa LeuSer Cys Glu Asn Lys Ile 65 70 75 80 Ile Ser Phe Lys Glu Met Asn Pro ProAsp Asn Ile Lys Asp Thr Lys 85 90 95 Ser Asp Ile Ile Phe Phe Gln Arg SerVal Pro Gly His Asp Asn Lys 100 105 110 Met Gln Phe Glu Ser Ser Ser TyrGlu Gly Tyr Phe Leu Ala Cys Glu 115 120 125 Lys Glu Arg Asp Leu Phe LysLeu Ile Leu Lys Lys Glu Asp Glu Leu 130 135 140 Gly Asp Arg Ser Ile MetPhe Thr Val Gln Asn Glu Asp 145 150 155 (2) INFORMATION FOR SEQ ID NO:27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 157 (B) TYPE: amino acid(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 27: Asn Phe Gly Arg Leu His Cys Thr Thr Ala ValIle Arg Asn Ile Asn 1 5 10 15 Asp Gln Val Leu Phe Val Asp Lys Arg GlnPro Val Phe Glu Asp Met 20 25 30 Thr Asp Ile Asp Gln Ser Ala Ser Glu ProGln Thr Arg Leu Ile Ile 35 40 45 Tyr Met Tyr Lys Asp Ser Glu Val Arg GlyLeu Ala Val Thr Leu Ser 50 55 60 Val Lys Asp Ser Lys Xaa Ser Thr Leu SerCys Lys Asn Lys Ile Ile 65 70 75 80 Ser Phe Glu Glu Met Asp Pro Pro GluAsn Ile Asp Asp Ile Gln Ser 85 90 95 Asp Leu Ile Phe Phe Gln Lys Arg ValPro Gly His Asn Lys Met Glu 100 105 110 Phe Glu Ser Ser Leu Tyr Glu GlyHis Phe Leu Ala Cys Gln Lys Glu 115 120 125 Asp Asp Ala Phe Lys Leu IleLeu Lys Lys Lys Asp Glu Asn Gly Asp 130 135 140 Lys Ser Val Met Phe ThrLeu Thr Asn Leu His Gln Ser 145 150 155 (2) INFORMATION FOR SEQ ID NO:28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: TCAGTCGACGCCACCATGAA TTGTAGAGAA 30 (2) INFORMATION FOR SEQ ID NO: 29: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 29: GAAGCGGCCG CATCATTAAG ACTCGGAAAGAAC 33 (2) INFORMATION FOR SEQ ID NO: 30: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 30: TCAGTCGACG CCACCATGAA TTGTAGAGAA TTA 33 (2)INFORMATION FOR SEQ ID NO: 31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQID NO: 31: GAAGCGGCCG CATCATTATC TTGTGAAGAC GTG 33 (2) INFORMATION FORSEQ ID NO: 32: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:TCAGTCGACG CCACCATGAA TTGTAGAG 28 (2) INFORMATION FOR SEQ ID NO: 33: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 53 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: GAAGCGGCCG CTCATTAGTGATGGTGATGG TGATGTGCAA CATGGTTAAG CCT 53 (2) INFORMATION FOR SEQ ID NO:34: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: TCAGTCGACGCCACCATGAA TTGTAGAG 28 (2) INFORMATION FOR SEQ ID NO: 35: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 53 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 35: GAAGCGGCCG CTCATTAGTG ATGGTGATGGTGATGTCTTT CAGTGAAACA GCT 53 (2) INFORMATION FOR SEQ ID NO: 36: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: TCAGTCGACG CCACCATGCATCATGAAGAA 30 (2) INFORMATION FOR SEQ ID NO: 37: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 51 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 37: GAAGCGGCCG CATCATTAGT GATGGTGATGGTGATGTGTA AAGACATGGC C 51

What is claimed is:
 1. A method for treating diseases associated withexcessive IL-18 induced immunoreaction, comprising: transformingeffector cells with an isolated DNA comprising a nucleotide sequenceencoding an IL-18 binding polypeptide capable of binding IL-18; andintroducing the transformed effector cells into a subject to provideadoptive immunogene therapy to treat diseases associated with excessiveIL-18 induced immunoreaction.
 2. The method according to claim 1,further comprising proliferating the transformed effector cells in vitroprior to introducing the transformed effector cells into a subject. 3.The method according to claim 2, wherein the effector cells are tumorcells collected from the subject to be treated.
 4. The method accordingto claim 2, wherein the effector cells are lymphocytes collected fromthe subject to be treated.
 5. A method for treating diseases associatedwith excessive IL-18 induced immunoreaction, comprising administering toa subject in need thereof an isolated DNA comprising a nucleotidesequence encoding an IL-18 binding polypeptide capable of binding IL-18.6. An isolated DNA comprising a nucleotide sequence encoding an IL-18binding polypeptide derived from an IL-18 receptor, wherein said IL-18receptor comprises the amino acid sequence of SEQ ID NO:
 21. 7. Theisolated DNA according to claim 6, wherein said nucleotide sequenceencoding said IL-18 binding polypeptide comprises a part or a whole ofthe nucleotide sequence of SEQ ID NO: 4 or a complementary sequencethereto.
 8. The isolated DNA according to claim 7, wherein saidnucleotide sequence encoding said IL-18 binding polypeptide is selectedfrom the group consisting of SEQ ID NO: 2, SEQ ID NO: 11, andcomplementary sequences thereto.